Apelin-13 polypeptide mutant

Abstract

Disclosed are an Apelin-13 polypeptide mutant, a pharmaceutical composition comprising the Apelin-13 polypeptide mutant, and a use thereof.

Description

Apelin-13 polypeptide mutants

[0001] Reference of Related Applications

[0002] The present disclosure claims the entire benefit of the Chinese Patent Application No. 202410906618.8, filed on July 5, 2024, entitled “Apelin-13 polypeptide mutants” and incorporates it by reference in its entirety.

[0003] TECHNICAL FIELD

[0004] The present disclosure generally relates to the field of biological medicine, and more particularly, to the field of polypeptides.

[0005] BACKGROUND

[0006] Cardiovascular disease is one of the major diseases that threaten human physical and mental health, and the prevalence rate is increasing year by year. Countries are facing major challenges in the prevention and treatment of cardiovascular disease, which has caused major social and economic burdens. Apelin receptor (APLNR) is a typical class A GPCR with important functions in the cardiovascular system, including promoting heart contraction, enhancing left ventricular stroke volume, vasodilation, diuresis, reducing systemic blood pressure, etc.

[0007] SUMMARY

[0008] In one aspect, the present disclosure relates to an Apelin-13 polypeptide mutant, comprising

[0009] (1) a polypeptide represented by general formula (I):

[0010] aa1-Arg-Pro-Arg-Leu-b1-His-cc1-Gly-Pro-DMet-Gly-Pro-Phe

[0011] General formula (I)

[0012] wherein b1 is a side chain negatively charged amino acid, aa1 is selected from C 11-25 hydrocarbyl-γGlu, carboxyl-C 10-24 hydrocarbyl-γGlu, C 11-25 hydrocarbyl-γGlu-mxOEG, carboxyl-C 10-24 hydrocarbyl-γGlu-mxOEG, C 11-25 hydrocarbyl-γGlu-nxPEG, carboxyl-C 10-24 hydrocarbyl-γGlu-nxPEG or PEG, cc1 is selected from C 11-25 hydrocarbyl-γGlu-Lys, carboxyl-C 10-24 hydrocarbyl-γGlu-Lys, C 11-25hydrocarbyl-mxOEG-glu-lys, carboxyl-c 10-24 hydrocarbyl-mxOEG-glu-lys or PEG-lys, and the amino acid residues at positions 1, 6 form an amide bond, wherein m is an integer from 1 to 5, n is an integer from 1 to 8; or

[0013] (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (I) and having the same function.

[0014] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0015] (1) a polypeptide represented by general formula (II):

[0016] aa2-cys-a2-pro-arg-leu-cys-his-lys-gly-pro-dmet-gly-pro-phe

[0017] general formula (II)

[0018] wherein a2 is selected from an amino acid having a hydrophobic side chain or an amino acid with a polar uncharged side chain, aa2 is selected from C 11-25 hydrocarbyl-glu, carboxyl-c 10-24 hydrocarbyl-glu, c 11-25 hydrocarbyl-glu-mxOEG, carboxyl-c 10-24 hydrocarbyl-glu-mxOEG, c 11-25 hydrocarbyl-glu-nxPEG, carboxyl-c 10-24 hydrocarbyl-glu-nxPEG or PEG, and the cysteine residues at positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5, n is an integer from 1 to 8; or

[0019] (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (II) and having the same function.

[0020] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0021] (1) a polypeptide represented by general formula (III-1):

[0022] aa3-cys-arg-a3-arg-b3-cys-his-c3-gly-pro-d3-e3-f3-g3

[0023] general formula (III-1)

[0024] wherein a3 is selected from an amino acid having a hydrophobic side chain or a polar uncharged amino acid, b3 is an amino acid having a hydrophobic side chain, c3 is an amino acid having a positively charged side chain, d3 and e3 are each independently selected from an amino acid having a hydrophobic side chain or Gly, f3 is an amino acid having a hydrophobic side chain or Pro, g3 is an amino acid having a hydrophobic side chain, aa3 is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or

[0025] (2) a polypeptide represented by general formula (III-2):

[0026] aa3’-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-a3’

[0027] general formula (III-2)

[0028] wherein a3’ is an amino acid having a hydrophobic side chain, aa3’ is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or

[0029] (3) a polypeptide having at least 70% homology to the polypeptide represented by general formula (III-1) or general formula (III-2) and having the same function.

[0030] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0031] (1) a polypeptide represented by general formula (IV):

[0032] aa4-Cys-Arg-Pro-a4-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0033] Formula (IV)

[0034] wherein a4 is an amino acid having a hydrophobic side chain, aa4 is selected from the group consisting of C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer of 1 to 5, and n is an integer of 1 to 8; or

[0035] (2) a polypeptide having at least 70% homology with the polypeptide represented by Formula (IV) and having the same function.

[0036] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant, which comprises

[0037] (1) a polypeptide represented by Formula (V-1):

[0038] aa5-Cys-Arg-a5-Arg-b5-Cys-His-c5-Gly-Pro-d5-e5-f5-g5

[0039] Formula (V-1)

[0040] wherein a5 is selected from the group consisting of an amino acid having a hydrophobic side chain or Pro, b5 is an amino acid having a hydrophobic side chain, c5 is an amino acid having a positively charged side chain, d5 and e5 are each independently selected from the group consisting of an amino acid having a hydrophobic side chain or Gly, f5 is an amino acid having a hydrophobic side chain or Pro, g5 is an amino acid having a hydrophobic side chain, aa5 is selected from the group consisting of C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer of 1 to 5, and n is an integer of 1 to 8; or

[0041] (2) a polypeptide represented by general formula (V-2):

[0042] aa5'-Cys-Arg-a5'-Arg-b5'-Cys-His-c5'-Gly-Pro-Nal-Pro-d5'

[0043] General formula (V-2)

[0044] wherein a5' is selected from an amino acid having a hydrophobic side chain or Pro, b5' is an amino acid having a hydrophobic side chain, c5' is an amino acid having a positively charged side chain, d5' is an amino acid having a hydrophobic side chain, and aa5' is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or

[0045] (3) a polypeptide having at least 70% homology to the polypeptide represented by general formula (V-1) or general formula (V-2) and having the same function.

[0046] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0047] (1) a polypeptide represented by general formula (VII):

[0048] aa7-Cys-Arg-Pro-Arg-Leu-Cys-a7-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0049] General formula (VII)

[0050] wherein a7 is selected from an amino acid having a hydrophobic side chain or an amino acid having a polar uncharged side chain, and aa7 is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24hydrocarbyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8, or

[0051] (2) a polypeptide having at least 70% homology to the polypeptide represented by general formula (VII) and having the same function.

[0052] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0053] (1) a polypeptide represented by general formula (VIII-1):

[0054] aa8-Cys-Arg-a8-Arg-b8-Cys-His-c8-Gly-Pro-DMet-Gly-d8-e8

[0055] General formula (VIII-1)

[0056] wherein a8 is selected from an amino acid having a hydrophobic side chain or Pro, b8 is an amino acid having a hydrophobic side chain, c8 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain, d8 is an amino acid having a hydrophobic side chain or Pro, e8 is an amino acid having a hydrophobic side chain, and aa8 is selected from C 11-25 hydrocarbyl-γGlu, carboxyl-C 10-24 hydrocarbyl-γGlu, carboxyl-C 11-25 hydrocarbyl-γGlu-mxOEG, carboxyl-C 10-24 hydrocarbyl-γGlu-3xOEG, carboxyl-C 11-25 hydrocarbyl-γGlu-nxPEG, carboxyl-C 10-24 hydrocarbyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8, or

[0057] (2) a polypeptide represented by general formula (VIII-2):

[0058] aa8’-Cys-Arg-Pro-Arg-Trp-Cys-His-a8’-Gly-Pro-Nal-Pro-Phe

[0059] General formula (VIII-2)

[0060] wherein a8’ is an amino acid having a positively charged side chain, and aa8’ is selected from C 11-25 hydrocarbyl-γGlu, carboxyl-C 10-24 hydrocarbyl-γGlu, carboxyl-C 10-24 hydrocarbyl-γGlu-2xOEG, carboxyl-C 10-24Alkylacyl-γGlu-3xOEG, C 11-25 Alkylacyl-γGlu-nxPEG, carboxyl-C 10-24 Alkylacyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or

[0061] (3) a polypeptide represented by general formula (VIII-1) or a polypeptide having at least 70% homology with general formula (VIII-2) and having the same function.

[0062] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0063] (1) a polypeptide represented by general formula (IX):

[0064] aa9-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-a9-Pro-DMet-Gly-Pro-Phe

[0065] General formula (IX)

[0066] wherein a9 is a polar uncharged amino acid, aa9 is selected from C 11-25 Alkylacyl-γGlu, carboxyl-C 10-24 Alkylacyl-γGlu, C 11-25 Alkylacyl-γGlu-mxOEG, C 11-25 Alkylacyl-γGlu-mxOEG, C 11-25 Alkylacyl-γGlu-nxPEG, carboxyl-C 10-24 Alkylacyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or

[0067] (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (IX) and having the same function.

[0068] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0069] (1) a polypeptide represented by general formula (XI-1):

[0070] aa11-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11-Pro-Phe

[0071] General formula (XI-1)

[0072] wherein a11 is selected from Ala, Phe, Nal, DNal, DMet, Glu or Nle, aa11 is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8;

[0073] (2) a polypeptide represented by general formula (XI-2):

[0074] aa11'-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11'-b11'-Pro-Phe

[0075] general formula (XI-2)

[0076] wherein a11' is selected from an amino acid having a hydrophobic side chain or a polar uncharged side chain, His or Pro, b11' is selected from an amino acid having a hydrophobic side chain or Gly, and aa11' is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or

[0077] (3) a polypeptide having at least 70% homology to the polypeptide represented by general formula (XI-1) or general formula (XI-2) and having the same function.

[0078] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0079] (1) a polypeptide represented by general formula (XII):

[0080] aa12-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-a12-Pro-Phe

[0081] Formula (XII)

[0082] wherein a12 is selected from an amino acid having a positively charged side chain, an amino acid having a hydrophobic side chain, an amino acid having a polar uncharged side chain, or an amino acid having a negatively charged side chain, and aa12 is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or

[0083] (2) a polypeptide having at least 70% homology to the polypeptide represented by Formula (XII) and having the same function.

[0084] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0085] (1) a polypeptide represented by Formula (XIII):

[0086] aa13-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-a13-Phe

[0087] Formula (XIII)

[0088] wherein a12 is selected from an amino acid having a hydrophobic side chain, an amino acid having a polar uncharged side chain, an amino acid having a negatively charged side chain, or His, and aa13 is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or

[0089] (2) a polypeptide having at least 70% homology to the polypeptide represented by Formula (XIII) and having the same function.

[0090] In still another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0091] (1) a polypeptide represented by general formula (XIV):

[0092] aa14-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-a14

[0093] General formula (XIV)

[0094] wherein a14 is selected from the group consisting of an amino acid having a hydrophobic side chain, an amino acid having a polar uncharged side chain, an amino acid having a negatively charged side chain, His, or Nal, and aa14 is selected from the group consisting of C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or

[0095] (2) a polypeptide having at least 70% homology to the polypeptide represented by general formula (XIV) and having the same function.

[0096] In still another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0097] (1) a polypeptide represented by general formula (XV):

[0098] aa15-Cys-a15-b15-c15-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe

[0099] General formula (XV)

[0100] wherein a15 and b15 are each independently selected from the group consisting of an amino acid having a positively charged side chain or Pro, c15 is selected from the group consisting of an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain, and aa15 is selected from the group consisting of C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25hydrocarbyl-γGlu-nxPEG, carboxyl-C 10-24 hydrocarbyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 4 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or

[0101] (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (XVI) and having the same function.

[0102] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0103] (1) a polypeptide represented by general formula (XVI):

[0104] aa16-Cys-a16-b16-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe

[0105] General formula (XVI)

[0106] wherein a16 is selected from an amino acid with a positively charged side chain or Pro, b16 is selected from an amino acid with a hydrophobic side chain, an amino acid with a positively charged side chain or Pro, and aa16 is selected from C 11-25 hydrocarbyl-γGlu, carboxyl-C 10-24 hydrocarbyl-γGlu, C 11-25 hydrocarbyl-γGlu-mxOEG, carboxyl-C 10-24 hydrocarbyl-γGlu-mxOEG, C 11-25 hydrocarbyl-γGlu-nxPEG, carboxyl-C 10-24 hydrocarbyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 4 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or

[0107] (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (XVI) and having the same function.

[0108] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0109] (1) a polypeptide represented by general formula (XVI):

[0110] *-Arg-a17-b17-Leu-*-His-C17-Gly-Pro-DMet-Gly-Pro-Phe

[0111] General formula (XVI)

[0112] wherein a17 and b17 are each independently selected from proline or arginine, or a17 and / or b17 are absent, c17 is a positively charged side chain amino acid, * is a non-natural amino acid, and a ring structure is formed between the *; or

[0113] (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (XVII) and having the same function.

[0114] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0115] (1) a polypeptide represented by general formula (XVIII):

[0116] a18-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-b18-Pro-Phe

[0117] General formula (XVIII)

[0118] wherein a18 is selected from:

[0119] b18 is selected from an amino acid having a hydrophobic side chain or an amino acid having a negatively charged side chain, and the cysteine residues at positions 1 and 6 form a disulfide bond; or

[0120] (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (XVIII) and having the same function.

[0121] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0122] (1) a polypeptide represented by general formula (XIV):

[0123] a19-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-b19-c19-Pro-Phe

[0124] General formula (XIV)

[0125] wherein a19 is selected from:

[0126] b19 is selected from an amino acid having a hydrophobic side chain or an amino acid having a negatively charged side chain, c19 is selected as an amino acid having a hydrophobic side chain, and the cysteine residues at positions 1 and 6 form a disulfide bond; or

[0127] (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (XIV) and having the same function.

[0128] In yet another aspect, the present disclosure relates to a pharmaceutical composition comprising an Apelin-13 polypeptide mutant as described herein, and a pharmaceutically acceptable carrier, diluent or excipient.

[0129] In certain embodiments, the pharmaceutical composition described herein further comprises at least another active ingredient.

[0130] In certain embodiments, exemplary examples of the at least another active ingredient that can be used in the pharmaceutical composition of the present disclosure include, but are not limited to, active ingredients that act on GLP1R, GLP2R, GIPR, GCGR, FGF21R, NPY2R, or GDF15.

[0131] In certain embodiments, exemplary examples of at least another active ingredient that can be used in the pharmaceutical compositions of the present disclosure include, but are not limited to, Vurolenatide, exenatide, ROSE-010, GX-G6, orforglipron, FNP-120, UTMD-GLP-1, GLP-1 mimetics, VTC-G15, curaglutide, Exendin-4, P-11, CTRS-101, HD-7671, HTL-097, PGN-OB2, GL-0034, dulaglutide, semaglutide, enagoglutide, supalutai, rE-4, HS-20004, BPI-3016, ZT002, ECC-5004, TJ-103, SHR-2042, MDR-001, LPXT-007, Tirzepatide, CT-868, NNC-0090-2746, AMG133, CT-388, LY3493269, VK-2735, DA4-JC, HISHS-3001, HISHS-2001, LBT-6030, SCO-094, HS-20094, GMA106, HZ010, BGM0504, HZ012, XW003+XW017, THDBH120, BEBT-808, RAY-1225, Cotadutide, Pegapamodutide, BI-456906, Mazdutide / IBI362, efinopegdutide, Pemvidutide, NN-6177, ZP-2929, oxyntomodulin, G-49, AGM-217, JNJ-54728518, DA-1726, HM-14220, DD01, PB-718, TB001, SHR-1816, TB-001, YH-25724, HEC88473, AP-026, GLP-1-Fc-PYY, ZP-7570 (dapiglutide), HTL-30023, QL-1005, LY3437943 (retatrutide), HM15211, SAR441255, HISHS-3001, MWN-101, and DR10624.

[0132] In another aspect, the present disclosure relates to a method of treating or preventing a disease or condition responsive to agonism of the APLNR receptor, comprising administering to an individual in need of said method a therapeutically or prophylactically effective amount of an Apelin-13 polypeptide mutant described herein or a pharmaceutical composition described herein.

[0133] In yet another aspect, the present disclosure relates to the use of an Apelin-13 polypeptide mutant as described herein in the manufacture of a medicament for the treatment or prevention of a disease or condition responsive to agonism of the APLNR receptor.

[0134] In yet another aspect, the present disclosure relates to the use of an Apelin-13 polypeptide mutant as described herein in the manufacture of a medicament for the treatment or prevention of a disease or condition responsive to agonism of the APLNR receptor.

[0135] BRIEF DESCRIPTION OF DRAWINGS

[0136] Figure 1 is a structural diagram of the apelin-APLNR-Gil complex, and a result diagram of interaction analysis between ligand and receptor, wherein A: cryo-EM density map and molecular model of the apelin-APLNR-Gil complex, B: cryo-EM density map and molecular model of the polypeptide ligand apelin and MM07, C: dose-dependent curves of apelin and MM07 induced Gi protein dissociation (left) and β-arrestin recruitment (right).

[0137] Figure 2 is a result diagram of APLNR bias signaling mechanism analysis, wherein A: density map of apelin interacting with key amino acid residues in APLNR affecting bias, B: density map of MM07 interacting with key amino acid residues in APLNR affecting bias, C-F: G protein activation and β-arrestin recruitment detection of key amino acid mutations interacting with Ml 1 and F13, G: pattern diagram of Ml 1 and F13 interacting with “twin hotspots” on the receptor and peptide agonists.

[0138] Figure 3 shows the SDS-page characterization results of the fusion protein of the present disclosure.

[0139] Figure 4 shows the effects of different administration groups on the body weight and food intake of DIO mice, wherein A: weight loss quality change during administration, B: proportion of decreased body weight to original body weight, C: Q2D food intake change during administration, D: cumulative food intake statistics.

[0140] Figure 5 shows the effects of different administration groups on the muscle content change of DIO mice, wherein A: muscle content change during administration of different administration groups, B: proportion change of muscle content change to initial muscle content, C: statistical analysis of significant difference of muscle content change.

[0141] Figure 6 shows the effects of different administration groups on the fat content change of DIO mice, wherein A: fat content change during administration of different administration groups, B: proportion change of fat content change to initial fat content, C: statistical analysis of significant difference of fat content change.

[0142] Figure 7 shows the effect of different administration groups on the change in fat mass of DIO mice, wherein A: change in fat mass during administration of different administration groups, B: change in fat mass as a percentage of initial fat mass, C: statistical analysis of significant differences in change in fat mass.

[0143] Figure 8 shows the effect of different administration groups on the change in muscle mass of DIO mice, wherein A: change in muscle mass during administration of different administration groups, B: change in muscle mass as a percentage of initial fat mass.

[0144] Figure 9 shows the effect of different administration groups on the change in muscle mass / fat mass of DIO mice, wherein A: change in muscle mass / fat mass during administration of different administration groups, B: statistical analysis of significant differences in change in muscle mass / fat mass.

[0145] Figure 10 shows the effect of sequence 216 on the change in muscle and fat tissue weight after dissection of DIO mice, wherein A: statistical analysis of muscle tissue weight (g) after dissection, B: statistical analysis of fat tissue weight (g) after dissection, C: statistical analysis of change in muscle tissue mass / fat tissue mass after dissection.

[0146] Figure 11 shows the effect of sequence 216 on the body weight and food intake of DIO mice, wherein A: weight loss mass (g) and percentage of body weight loss (%) during administration, B: cumulative food intake statistics during administration.

[0147] Figure 12 shows the effect of sequence 216 on the weight of different fat tissues of DIO mice, wherein A: mass statistics of perirenal white fat rpWAT, B: epididymal white fat eWAT, C: inguinal white fat iWAT, D: brown fat BAT after dissection after 28 days of administration.

[0148] Figure 13 shows the TAC mouse model and administration scheme.

[0149] Figure 14 shows myocardial cell immunofluorescence.

[0150] Figure 15 shows the results of echocardiography detection of TAC model.

[0151] Figure 16 shows the results of myocardial cell hypertrophy detection.

[0152] Figure 17 shows the results of cardiac hypertrophy detection.

[0153] Figure 18 shows the results of cardiac hypertrophy-related factor level detection.

[0154] Figure 19 shows the results of myocardial fibrosis detection.

[0155] Detailed description

[0156] In the following description, certain specific details are set forth in order to provide a thorough understanding of various disclosed implementations. However, one skilled in the relevant art will recognize that implementations can be practiced without one or more of the specific details, or with other methods, components, materials, etc.

[0157] Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise," "comprising," and the like are to be construed in an open, inclusive sense, as

[0158] As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0159] References in the specification to "one embodiment," "another embodiment," or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase "in one embodiment" or "in an embodiment" in various places in the specification are not necessarily all referring to the same embodiment.

[0160] It should be understood that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0161] Definitions

[0162] Thus, unless stated to the contrary, the following terms used in the description and associated claims have the following meanings:

[0163] The abbreviations denoting certain chemical moieties named in the disclosure indicate the total number of carbon atoms present in the indicated chemical moiety. For example, C 1-4 Alkyl describes an alkyl group having from 1 to 4 carbon atoms in the whole, as defined below, while C3-C6alkyl describes an alkyl group having from 3 to 6 carbon atoms in the whole. 10 Cycloalkyl describes a cycloalkyl group having from 3 to 10 carbon atoms in the whole, as defined below. The total number of carbons in the abbreviation does not include carbons that can be present in substituents of the group.

[0164] In this disclosure, the term "alkyl group" refers to an aliphatic hydrocarbon group. The alkyl group can be a "saturated alkyl group," meaning it does not contain any alkene or alkyne portion, i.e., an alkyl group. The alkyl group can also be an "unsaturated alkyl group," meaning it contains at least one alkene or alkyne portion. An "alkene" portion, i.e., an alkenyl group, refers to a straight-chain or branched hydrocarbon chain group consisting of 12 to 24 carbon atoms and at least one carbon-carbon double bond, connected to the rest of the molecule by single bonds, such as vinyl, propenyl, butenyl, pentenyl, pent-1,4-dienyl, etc. An "alkynyl" portion refers to a straight-chain or branched hydrocarbon chain group consisting of 12 to 24 carbon atoms and at least one carbon-carbon triple bond, connected to the rest of the molecule by single bonds. The alkyl group, whether saturated or unsaturated, can be branched or straight-chain.

[0165] The hydrocarbon group may have 12 to 24 carbon atoms (whenever it appears in this disclosure, a numerical range such as “12 to 14” refers to every integer in the given range; such as “12 to 24” means that the hydrocarbon group may have 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, etc. up to and including 24 carbon atoms, although this definition also covers the occurrence of the term “hydrocarbon” where no numerical range is specified).

[0166] In this disclosure, the term "C" 11-25 "Hydroyl-γGlu" refers to a group having the following structure:

[0167] In this disclosure, the term "carboxyl-C" is used. 10-24 "Hydroyl-γGlu" refers to a group having the following structure:

[0168] In this disclosure, the term "C" 11-25 "Hydroylacyl-γGlu-m×OEG" refers to a group having the following structure:

[0169] In this disclosure, the term "carboxyl-C" is used. 10-24 "Hydroylacyl-γGlu-m×OEG" refers to a group having the following structure:

[0170] In this disclosure, the term "C" 11-25 "Hydroyl-γGlu-n×PEG" refers to a group having the following structure:

[0171] In this disclosure, the term "carboxyl-C" is used. 10-24 "Hydroyl-γGlu-n×PEG" refers to a group having the following structure:

[0172] In the present disclosure, the term "C 11-25 Heteroaryl-γGlu-Lys" refers to a group having the following structure:

[0173] In the present disclosure, the term "Carboxyl-C 10-24 Heteroaryl-γGlu-Lys" refers to a group having the following structure:

[0174] In the present disclosure, the term "PEG" refers to a group having the following structure:

[0175] n = 2-10

[0176] In the present disclosure, wild-type Apelin-13 polypeptide refers to a polypeptide having the following amino acid sequence: Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe, which is denoted by single letter as QRPRLSHKGPMPF.

[0177] In the present disclosure, the term "Cysteine residues at positions 1, 6 form disulfide bond" refers to the formation of disulfide bond by the cysteine residues at positions 1, 6 of the Apelin-13 polypeptide mutants of the present disclosure corresponding to the wild-type Apelin-13 polypeptide.

[0178] In the present disclosure, the term "Disulfide bridge: a-b (disulfide bond at positions a, b)" refers to the formation of disulfide bond by the Apelin-13 polypeptide mutants of the present disclosure at positions a, b corresponding to the wild-type Apelin-13 polypeptide.

[0179] In the present disclosure, the term "Disulfide bridge: 1-6 (disulfide bond at positions 1, 6)" refers to the formation of disulfide bond by the Apelin-13 polypeptide mutants of the present disclosure at positions 1, 6 corresponding to the wild-type Apelin-13 polypeptide.

[0180] In the present disclosure, the term "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening, diluting, preserving, dye / colorant, flavor-enhancing, surface-active, wetting, dispersing, suspending, stabilizing, isotonic, solvent, or emulsifying agent, etc., which has been approved by the U.S. Food and Drug Administration and can be used in humans or animals without side effects on the components of the pharmaceutical composition in various forms of carriers.

[0181] In the present disclosure, the term "therapeutically effective amount" means an amount of a compound or combination of compounds that ameliorates, attenuates or eliminates a particular disease or condition and symptoms of a particular disease or condition, or that prevents or delays the onset of a particular disease or condition or symptoms of a particular disease or condition. The amount of a compound described in the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, and the age, weight, etc. of the mammal to be treated, but can be routinely determined by the skilled artisan according to his own knowledge and the present disclosure.

[0182] "treating" or "treatment" as used in the present disclosure encompasses treatment of a related disease or disorder in a mammal, e.g., in a human, having the related disease or disorder and includes:

[0183] (i) preventing a disease or condition from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it;

[0184] (ii) inhibiting the disease or condition, i.e., arresting its development; or

[0185] (iii) relieving the disease or condition, i.e., causing the disease or condition to regress or not progress.

[0186] As used in the present disclosure, the terms "disease" and "condition" can be used interchangeably or can be different in that a particular disease or condition can not have a known causative agent (and thus cannot be explained in terms of etiology) and thus it is not recognized as a disease but rather as an undesirable condition or disorder for which a more or less specific series of symptoms has been identified by clinicians. DETAILED DESCRIPTION

[0187] In one aspect, the present disclosure relates to Apelin-13 polypeptide mutants comprising

[0188] (1) a polypeptide according to general formula (I):

[0189] aa1-Arg-Pro-Arg-Leu-b1-His-cc1-Gly-Pro-DMet-Gly-Pro-Phe

[0190] General formula (I)

[0191] wherein b1 is a side chain negatively charged amino acid and aa1 is selected from the group consisting of C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24Alkylacyl-γGlu-mxOEG, C 11-25 Alkylacyl-γGlu-nxPEG, carboxyl-C 10-24 Alkylacyl-γGlu-nxPEG or PEG, cc1 is selected from C 11-25 Alkylacyl-γGlu-Lys, C 11-25 Alkylacyl-γGlu-Lys, C 11-25 Alkylacyl-mxOEG-γGlu-Lys, C 11-25 Alkylacyl-mxOEG-γGlu-Lys or PEG-Lys, and the amino acid residues at positions 1, 6 form an amide bond, wherein m is an integer of 1 to 5, and n is an integer of 1 to 8; or

[0192] (2) a polypeptide having at least 70% homology with the polypeptide represented by the general formula (I) and having the same function.

[0193] In certain embodiments, exemplary examples of a1 that can be used in the present disclosure include, but are not limited to, Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gin, Cys, Sec, Gly, Pro, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.

[0194] In certain embodiments, exemplary examples of b1 that can be used in the present disclosure include, but are not limited to, Glu and Asp.

[0195] In certain embodiments, exemplary examples of aa1 that can be used in the present disclosure include, but are not limited to, C 11-25 Alkylacyl-γGlu, carboxyl-C 10-24 Alkylacyl-γGlu, carboxyl-C 10-24 Alkylacyl-γGlu-2xOEG, carboxyl-C 10-24 Alkylacyl-γGlu-3xOEG or PEG.

[0196] In certain embodiments, exemplary examples of cc1 that can be used in the present disclosure include, but are not limited to, C 11-25 Alkylacyl-γGlu, carboxyl-C 10-24 Alkylacyl-γGlu, carboxyl-C 10-24 Alkylacyl-γGlu-2xOEG, carboxyl-C 10-24 Alkylacyl-γGlu-3xOEG or PEG.

[0197] In certain embodiments, the Apelin-13 polypeptide mutant of the present disclosure has a mutation at M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55Y264 6.51 K265 6.55 Y35 1.39 T295 7.39 Y299 7.49 F78 2.53 W85 2.60 Y88 2.63 Y93 ECL1 M113 3.36 R168 4.64 Y185 ECL2 E198 5.35 Y271 6.58 S275 6.62 M288 7.32 F291 7.35 P163 4.59 L201 5.38 V164 4.60 S205 5.42 P292 7.36 M288 7.32 Q180 ECL2 or Y182 ECL2 in combination.

[0198] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 F110 3.33 I109 3.32 K268 6.55 Y264 6.51 K265 6.55 Y35 1.39 T295 7.39 or Y299 7.49 in combination, thereby affecting G protein bias.

[0199] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide represented by the general formula (I).

[0200] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are:

[0201] C 15 alkyl-CO-yGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (amide bond at positions 1,6).

[0202] In another aspect, the present disclosure relates to Apelin-13 polypeptide mutants comprising

[0203] (1) a polypeptide represented by general formula (II):

[0204] aa2-Cys-a2-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0205] General Formula (II)

[0206] wherein a2 is selected from an amino acid having a hydrophobic side chain or an amino acid having a polar uncharged side chain, and aa2 is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer of 1 to 5, and n is an integer of 1 to 8; or

[0207] (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (II) and having the same function.

[0208] In certain embodiments, exemplary examples of a2 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, and Gin.

[0209] In certain embodiments, exemplary examples of aa2 that can be used in the present disclosure include, but are not limited to, C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 10-24 alkanoyl-γGlu-2xOEG, carboxyl-C 10-24 alkanoyl-γGlu-3xOEG or PEG.

[0210] In certain embodiments, the Apelin-13 polypeptide mutant of the present disclosure has a mutation at M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60, Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 in combination.

[0211] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 or Y299 7.49 in combination, thereby affecting G protein bias.

[0212] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide represented by the general formula (II).

[0213] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from:

[0214] C 19 alkyl-CO-yGlu-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0215] C 17 alkyl-CO-yGlu-2xOEG-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0216] COOHC 18Alkyl-CO-γGlu-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0217] COOHC 22 Alkyl-CO-γGlu-2xOEG-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0218] PEG2-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0219] PEG3-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0220] PEG5-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and

[0221] PEG8-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

[0222] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0223] (1) a polypeptide represented by the general formula (III-1):

[0224] aa3-Cys-Arg-a3-Arg-b3-Cys-His-c3-Gly-Pro-d3-e3-f3-g3

[0225] General formula (III-1)

[0226] wherein a3 is selected from an amino acid having a hydrophobic side chain or a polar uncharged amino acid, b3 is an amino acid having a hydrophobic side chain, c3 is an amino acid having a positively charged side chain, d3 and e3 are each independently selected from an amino acid having a hydrophobic side chain or Gly, f3 is an amino acid having a hydrophobic side chain or Pro, g3 is an amino acid having a hydrophobic side chain, aa3 is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or

[0227] (2) a polypeptide represented by General Formula (III-2):

[0228] aa3'-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-a3'

[0229] General Formula (III-2)

[0230] wherein a3' is an amino acid having a hydrophobic side chain, aa3' is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or

[0231] (3) a polypeptide having at least 70% homology to the polypeptide represented by General Formula (III-1) or General Formula (III-2) and having the same function.

[0232] In certain embodiments, exemplary examples of a3 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, and Gin.

[0233] In certain embodiments, exemplary examples of b3 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

[0234] In certain embodiments, exemplary examples of c3 that can be used in the present disclosure include, but are not limited to, Lys and Arg.

[0235] In certain embodiments, exemplary examples of d3 and e3 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, DMet, Phe, Tyr, Trp, and Gly.

[0236] In certain embodiments, exemplary examples of f3 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, and Pro.

[0237] In certain embodiments, exemplary examples of g3 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

[0238] In certain embodiments, exemplary examples of aa3 that can be used in the present disclosure include, but are not limited to, C 11-25 Alkylacyl-γGlu, carboxy-C 10-24 Alkylacyl-γGlu, carboxy-C 10-24 Alkylacyl-γGlu-2xOEG, carboxy-C 10-24 Alkylacyl-γGlu-3xOEG or PEG.

[0239] In certain embodiments, exemplary examples of a3' that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

[0240] In certain embodiments, exemplary examples of aa3' that can be used in the present disclosure include, but are not limited to, C 11-25 Alkylacyl-γGlu, carboxy-C 10-24 Alkylacyl-γGlu, carboxy-C 10-24 Alkylacyl-γGlu-2xOEG, carboxy-C 10-24 Alkylacyl-γGlu-3xOEG or PEG.

[0241] In certain embodiments, Apelin-13 polypeptide mutants of the present disclosure have M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K2656.55 Y35 1.39 T295 7.39 Y299 7.49 F78 2.53 W85 2.60 Y88 2.63 Y93 ECL1 M113 3.36 R168 4.64 Y185 ECL2 E198 5.35 Y271 6.58 S275 6.62 M288 7.32 F291 7.35 P163 4.59 L201 5.38 V164 4.60 S205 5.42 P292 7.36 M288 7.32 Q180 ECL2 or Y182 ECL2 in combination.

[0242] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 F110 3.33 I109 3.32 K268 6.55 Y264 6.51 K265 6.55 Y35 1.39 T295 7.39 or Y299 7.49 in combination, thereby affecting G protein bias.

[0243] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide according to general formula (III-1) or a polypeptide according to general formula (III-2).

[0244] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from:

[0245] C 15 alkyl-CO-yGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Leu (Disulfide bridge: 1-6);

[0246] C 15Alkyl-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Tyr-Phe (Disulfide bridge: 1-6);

[0247] C 15 Alkyl-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Tyr-Phe (Disulfide bridge: 1-6);

[0248] C 15 Alkyl-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Tyr-Phe (Disulfide bridge: 1-6);

[0249] C 15 Alkyl-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Tyr-Phe (Disulfide bridge: 1-6);

[0250] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0251] (1) a polypeptide represented by general formula (IV):

[0252] aa4-Cys-Arg-Pro-a4-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0253] General formula (IV)

[0254] wherein a4 is an amino acid having a hydrophobic side chain, aa3 is selected from C 11-25 Alkanoyl-γGlu, carboxyl-C 10-24 Alkanoyl-γGlu, C 11-25 Alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 Acyl-γGlu-mxOEG, C 11-25 Alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 Alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5, n is an integer from 1 to 8; or

[0255] (2) polypeptides having at least 70% homology to the polypeptides represented by general formula (IV) and having the same function.

[0256] In certain embodiments, exemplary examples of a4 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

[0257] In certain embodiments, exemplary examples of aa4 that can be used in the present disclosure include, but are not limited to, C 11-25 hydrocarbyl-gamma Glu, carboxy-C 10-24 hydrocarbyl-gamma Glu, carboxy-C 10-24 hydrocarbyl-gamma Glu-2xOEG, carboxy-C 10-24 hydrocarbyl-gamma Glu-3xOEG or PEG.

[0258] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , 180 ECL2 , or 182 ECL2 .

[0259] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55Y264 6.51 K265 6.55 Y35 1.39 T295 7.39 or Y299 7.49 bind, thereby influencing G protein bias.

[0260] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide represented by general formula (IV).

[0261] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from:

[0262] C 19 alkyl-CO-yGlu-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0263] C 17 alkyl-CO-yGlu-2xOEG-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0264] COOHC 18 alkyl-CO-yGlu-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0265] COOHC 22 alkyl-CO-yGlu-2xOEG-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0266] PEG2-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0267] PEG3-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0268] PEG5-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and

[0269] PEG8-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe.

[0270] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0271] (1) a polypeptide represented by general formula (V-1):

[0272] aa5-Cys-Arg-a5-Arg-b5-Cys-His-c5-Gly-Pro-d5-e5-f5-g5

[0273] General formula (V-1)

[0274] wherein a5 is selected from an amino acid having a hydrophobic side chain or Pro, b5 is an amino acid having a hydrophobic side chain, c5 is an amino acid having a positively charged side chain, d5 and e5 are each independently selected from an amino acid having a hydrophobic side chain or Gly, f5 is an amino acid having a hydrophobic side chain or Pro, g5 is an amino acid having a hydrophobic side chain, aa1 is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or

[0275] (2) a polypeptide represented by general formula (V-2):

[0276] aa5’-Cys-Arg-a5’-Arg-b5’-Cys-His-c5’-Gly-Pro-Nal-Pro-d5’

[0277] General formula (V-2)

[0278] wherein a5' is selected from an amino acid having a hydrophobic side chain or Pro, b5' is an amino acid having a hydrophobic side chain, c5' is an amino acid having a positively charged side chain, d5' is an amino acid having a hydrophobic side chain, aa5' is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or

[0279] (3) a polypeptide represented by the general formula (V-1) or a polypeptide having at least 70% homology with the general formula (V-2) and having the same function.

[0280] In certain embodiments, exemplary examples of a5 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, and Pro.

[0281] In certain embodiments, exemplary examples of b5 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

[0282] In certain embodiments, exemplary examples of c5 that can be used in the present disclosure include, but are not limited to, Lys and Arg.

[0283] In certain embodiments, exemplary examples of d5 and e5 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, DMet, Phe, Tyr, Trp, and Gly.

[0284] In certain embodiments, exemplary examples of f5 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, and Pro.

[0285] In certain embodiments, exemplary examples of g5 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

[0286] In certain embodiments, exemplary examples of aa5 that can be used in the present disclosure include, but are not limited to, C 11-25 alkyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, carboxyl-C10-24 Alkylacyl-γGlu-2xOEG, carboxy-C 10-24 Alkylacyl-γGlu-3xOEG or PEG.

[0287] In certain embodiments, exemplary examples of a5’ that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, and Pro.

[0288] In certain embodiments, exemplary examples of b5’ that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

[0289] In certain embodiments, exemplary examples of c5’ that can be used in the present disclosure include, but are not limited to, Lys and Arg.

[0290] In certain embodiments, exemplary examples of d5’ that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

[0291] In certain embodiments, exemplary examples of aa5’ that can be used in the present disclosure include, but are not limited to, C 11-25 Alkylacyl-γGlu, carboxy-C 10-24 Alkylacyl-γGlu, carboxy-C 10-24 Alkylacyl-γGlu-2xOEG, carboxy-C 10-24 Alkylacyl-γGlu-3xOEG or PEG.

[0292] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure have a M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F2917.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 in combination.

[0293] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 or Y299 7.49 in combination, thereby affecting G protein bias.

[0294] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide according to general formula (V-1) or a polypeptide according to general formula (V-2).

[0295] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from:

[0296] C 17 alkyl-CO-yGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0297] C 19 alkyl-CO-yGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0298] C 21 alkyl-CO-yGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0299] COOHC 16Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0300] COOHC 18 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0301] C 17 Alkyl-CO-γGlu-OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0302] C 17 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0303] C 17 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0304] C 11 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0305] C 13 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0306] C 15Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0307] C 19 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0308] C 21 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0309] COOHC 16 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0310] C 17 Alkyl-CO-γGlu-4xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0311] C 17 Alkyl-CO-γGlu-5xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe C17alkCO (Disulfide bridge: 1-6);

[0312] C 19 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe (Disulfide bridge: 1-6);

[0313] C 17Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro- Phe (Disulfide bridge: 1-6); and

[0314] PEG8-Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe (Disulfide bridge: 1-6).

[0315] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0316] (1) a polypeptide represented by general formula (VII):

[0317] aa7-Cys-Arg-Pro-Arg-Leu-Cys-a7-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0318] General formula (VII)

[0319] wherein a7 is selected from an amino acid having a hydrophobic side chain or a polar uncharged side chain, and aa7 is selected from C 11-25 Alkanoyl-γGlu, carboxyl-C 10-24 Alkanoyl-γGlu, C 11-25 Alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 Alkanoyl-γGlu-mxOEG, C 11-25 Alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 Alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or

[0320] (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (VII) and having the same function.

[0321] In certain embodiments, exemplary examples of a7 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, and Gin.

[0322] In certain embodiments, exemplary examples of aa7 that can be used in the present disclosure include, but are not limited to, C 11-25 Alkanoyl-γGlu, carboxyl-C 10-24 Alkanoyl-γGlu, C 10-24hydrocarbylacyl-γGlu-2xOEG, carboxy-C 10-24 hydrocarbylacyl-γGlu-3xOEG or PEG.

[0323] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 F110 3.33 I109 3.32 K268 6.55 Y264 6.51 K265 6.55 Y35 1.39 T295 7.39 Y299 7.49 F78 2.53 W85 2.60 Y88 2.63 Y93 ECL1 M113 3.36 R168 4.64 Y185 ECL2 E198 5.35 Y271 6.58 S275 6.62 M288 7.32 F291 7.35 P163 4.59 L201 5.38 V164 4.60 S205 5.42 P292 7.36 M288 7.32 Q180 ECL2 or Y182 ECL2 .

[0324] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 F110 3.33 I109 3.32 K268 6.55 Y264 6.51 K265 6.55 Y35 1.39 T295 7.39 or Y299 7.49 , thereby affecting G protein bias.

[0325] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide represented by the general formula (VII).

[0326] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from:

[0327] C 19 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0328] C 17 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0329] COOH 18 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0330] COOH 22 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0331] PEG2-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0332] PEG3-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0333] PEG5-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and

[0334] PEG8-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

[0335] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0336] (1) a polypeptide represented by General Formula (VIII-1):

[0337] aa8-Cys-Arg-a8-Arg-b8-Cys-His-c8-Gly-Pro-DMet-Gly-d8-e8

[0338] General Formula (VIII-1)

[0339] wherein a8 is selected from an amino acid having a hydrophobic side chain or Pro, b8 is an amino acid having a hydrophobic side chain, c8 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain, d8 is an amino acid having a hydrophobic side chain or Pro, e8 is an amino acid having a hydrophobic side chain, and aa8 is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer of 1 to 5, and n is an integer of 1 to 8; or

[0340] (2) a polypeptide represented by General Formula (VIII-2):

[0341] aa8’-Cys-Arg-Pro-Arg-Trp-Cys-His-a8’-Gly-Pro-Nal-Pro-Phe

[0342] General Formula (VIII-2)

[0343] wherein a8’ is an amino acid having a positively charged side chain, and aa8’ is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer of 1 to 5, and n is an integer of 1 to 8; or

[0344] (3) a polypeptide represented by the general formula (VIII-1) or a polypeptide having at least 70% homology with the general formula (VIII-2) and having the same function.

[0345] In certain embodiments, exemplary examples of a8 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, and Pro.

[0346] In certain embodiments, exemplary examples of b8 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

[0347] In certain embodiments, exemplary examples of c8 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Arg, and Lys.

[0348] In certain embodiments, exemplary examples of d8 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, and Pro.

[0349] In certain embodiments, exemplary examples of e8 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

[0350] In certain embodiments, exemplary examples of aa8 that can be used in the present disclosure include, but are not limited to, C 11-25 hydrocarbyl-gammaGlu, carboxy-C 10-24 hydrocarbyl-gammaGlu, carboxy-C 10-24 hydrocarbyl-gammaGlu-2xOEG, carboxy-C 10-24 hydrocarbyl-gammaGlu-3xOEG or PEG.

[0351] In certain embodiments, exemplary examples of a8’ that can be used in the present disclosure include, but are not limited to, Arg and Lys.

[0352] In certain embodiments, exemplary examples of aa8’ that can be used in the present disclosure include, but are not limited to, C 11-25 hydrocarbyl-gammaGlu, carboxy-C 10-24 hydrocarbyl-gammaGlu, carboxy-C 10-24 hydrocarbyl-gammaGlu-2xOEG, carboxy-C 10-24 hydrocarbyl-gammaGlu-3xOEG or PEG.

[0353] In certain embodiments, the Apelin-13 polypeptide mutant of the present disclosure has an M183 ECL2F110 3.33 I109 3.32 K268 6.55 Y264 6.51 K265 6.55 Y35 1.39 T295 7.39 Y299 7.49 F78 2.53 W85 2.60 Y88 2.63 Y93 ECL1 M113 3.36 R168 4.64 Y185 ECL2 E198 5.35 Y271 6.58 S275 6.62 M288 7.32 F291 7.35 P163 4.59 L201 5.38 V164 4.60 S205 5.42 P292 7.36 M288 7.32 Q180 ECL2 or Y182 ECL2 in combination.

[0354] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 F110 3.33 I109 3.32 K268 6.55 Y264 6.51 K265 6.55 Y35 1.39 T295 7.39 or Y299 7.49 in combination, thereby affecting G protein bias.

[0355] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide according to general formula (VIII-1) or a polypeptide according to general formula (VIII-2).

[0356] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from the group consisting of:

[0357] C 15Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0358] C 17 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0359] C 19 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0360] C 21 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0361] COOHC 16 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0362] COOHC 18 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0363] C 17 Alkyl-CO-γGlu-OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0364] C 17Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0365] C 17 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0366] C 11 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0367] C 13 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0368] C 15 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0369] C 19 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0370] C 21 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0371] COOHC 16Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0372] COOH-C 18 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0373] C 17 Alkyl-CO-γGlu-4xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0374] C 17 Alkyl-CO-γGlu-5xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0375] C 17 Alkyl-CO-3xOEG-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0376] C 17 Alkyl-CO-DγGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0377] C 17 Alkyl-CO-γGlu-8PEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0378] C 17Alkyl-CO-2xγGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0379] C 17 Alkyl-CO-3xγGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0380] Phenyl-C 17 Alkyl-CO-PAMBA-βAla-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0381] C 17 Alkyl-CO-Abu-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0382] C 17 Alkyl-CO-Abu-2xγGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0383] C 17 Alkyl-CO-Abu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0384] COOHC 12 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0385] COOHC 14Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0386] COOHC 16 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0387] COOHC 18 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0388] COOHC 20 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0389] COOHC 22 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0390] COOHC 18 Alkyl-CO-γGlu-4xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0391] COOHC 18 Alkyl-CO-2xOEG-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0392] COOHC 18Alkyl-CO-Abu-2xγGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0393] COOHC 18 Alkyl-CO-Abu-2xγGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0394] COOHC 18 Alkyl-CO-Abu-2xγGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0395] COOHC 18 Alkyl-CO-Abu-2xγGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0396] COOHC 18 Alkyl-CO-Abu-2xγGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0397] COOHC 18 Alkyl-CO-Abu-2xγGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0398] COOHC 18 Alkyl-CO-Abu-2xγGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0399] COOHC 18Alkyl-CO-γGlu-5xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0400] COOHC 18 Alkyl-CO-γGlu-5xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0401] COOHC 18 Alkyl-CO-γGlu-5xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0402] COOHC 18 Alkyl-CO-γGlu-5xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0403] COOHC 16 Alkyl-CO-γGlu-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6);

[0404] COOHC 18 Alkyl-CO-γGlu-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6);

[0405] COOHC 22 Alkyl-CO-γGlu-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6);

[0406] COOHC16 Alkyl-CO-γGlu-2xOEG-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 1-6);

[0407] COOHC 18 Alkyl-CO-γGlu-2xOEG-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 1-6);

[0408] COOHC 22 Alkyl-CO-γGlu-2xOEG-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 1-6);

[0409] COOHC 22 Alkyl-CO-γGlu-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 1-6); and

[0410] COOHC 22 Alkyl-CO-γGlu-2xOEG-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 1-6).

[0411] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0412] (1) a polypeptide represented by the general formula (IX):

[0413] aa9-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-a9-Pro-DMet-Gly-Pro-Phe

[0414] General formula (IX)

[0415] wherein a9 is a polar uncharged amino acid, aa9 is selected from the group consisting of C 11-25 Alkylacyl-γGlu, carboxyl-C 10-24 Alkylacyl-γGlu, C 11-25 Alkylacyl-γGlu-mxOEG, carboxyl-C 10-24 Acyl-γGlu-mxOEG, C 11-25 Alkylacyl-γGlu-nxPEG, carboxyl-C 10-24 Alkylacyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or

[0416] (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (IX) and having the same function.

[0417] In certain embodiments, exemplary examples of a9 that can be used in the present disclosure include, but are not limited to, Ser, Thr, Asn, and Gin.

[0418] In certain embodiments, exemplary examples of aa9 that can be used in the present disclosure include, but are not limited to, C 11-25 Alkylacyl-γGlu, carboxyl-C 10-24 Alkylacyl-γGlu, carboxyl-C 10-24 Alkylacyl-γGlu-2xOEG, carboxyl-C 10-24 Alkylacyl-γGlu-3xOEG or PEG.

[0419] In certain embodiments, the Apelin-13 polypeptide mutant of the present disclosure has a substitution at M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L2015.38 V164 4.60 S205 5.42 P292 7.36 M288 7.32 Q180 ECL2 or Y182 ECL2 bind.

[0420] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 F110 3.33 I109 3.32 K268 6.55 Y264 6.51 K265 6.55 Y35 1.39 T295 7.39 or Y299 7.49 and thereby affect G protein bias.

[0421] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide represented by general formula (IX).

[0422] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are:

[0423] C 19 alkyl-CO-yGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0424] C 17 alkyl-CO-yGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0425] COOHC 18 alkyl-CO-yGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0426] COOHC 22Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0427] PEG2-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0428] PEG3-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0429] PEG5-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and

[0430] PEG8-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

[0431] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0432] (1) a polypeptide represented by general formula (XI-1):

[0433] aa11-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11-Pro-Phe

[0434] General formula (XI-1)

[0435] wherein a11 is selected from the group consisting of Ala, Phe, Nal, DNal, DMet, Glu or Nle, and aa11 is selected from the group consisting of C 11-25 Alkyl-CO-γGlu, Carboxyl-C 10-24 Alkyl-CO-γGlu, C 11-25 Alkyl-CO-γGlu-mxOEG, C 11-25 Alkyl-CO-γGlu-mxOEG, C 11-25 Alkyl-CO-γGlu-nxPEG, Carboxyl-C 10-24hydrocarbonyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or

[0436] (2) a polypeptide represented by General Formula (XI-2):

[0437] aa11'-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11'-b11'-Pro-Phe

[0438] General Formula (XI-2)

[0439] wherein a11' is selected from an amino acid having a hydrophobic side chain or a polar uncharged side chain, His, or Pro, b11' is selected from an amino acid having a hydrophobic side chain or Gly, and aa11' is selected from C 11-25 hydrocarbonyl-γGlu, carboxyl-C 10-24 hydrocarbonyl-γGlu, carboxyl-C 11-25 hydrocarbonyl-γGlu-2xOEG, carboxyl-C 10-24 hydrocarbonyl-γGlu-3xOEG or PEG. 11-25 hydrocarbonyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or 10-24 hydrocarbonyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or

[0440] (3) a polypeptide having at least 70% homology to the polypeptide represented by General Formula (XI-1) or General Formula (XI-2) and having the same function.

[0441] In certain embodiments, exemplary examples of a11' that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gin, His, and Pro.

[0442] In certain embodiments, exemplary examples of b11' that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, and Gly.

[0443] In certain embodiments, exemplary examples of aa11that can be used in the present disclosure include, but are not limited to, C 11-25 hydrocarbonyl-γGlu, carboxyl-C 10-24 hydrocarbonyl-γGlu, carboxyl-C 10-24 hydrocarbonyl-γGlu-2xOEG, carboxyl-C 10-24 hydrocarbonyl-γGlu-3xOEG or PEG.

[0444] In certain embodiments, exemplary examples of aa11’ that can be used in the present disclosure include, but are not limited to, C 11-25 hydrocarbyl-ylGlu, carboxy-C 10-24 hydrocarbyl-ylGlu, carboxy-C 10-24 hydrocarbyl-ylGlu-2xOEG, carboxy-C 10-24 hydrocarbyl-ylGlu-3xOEG or PEG.

[0445] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

[0446] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , or Y299 7.49 , thereby affecting G protein bias.

[0447] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide according to general formula (XI-1) or a polypeptide according to general formula (XI-2).

[0448] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from the group consisting of:

[0449] C 15 alkyl-CO-yGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Nal-Pro-Phe (Disulfide bridge: 1-6);

[0450] C 19 alkyl-CO-yGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6);

[0451] C 17 alkyl-CO-yGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6);

[0452] COOH 18 alkyl-CO-yGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6);

[0453] COOH 22 alkyl-CO-yGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6);

[0454] PEG2-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6);

[0455] PEG3-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6);

[0456] PEG5-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6); and

[0457] PEG8-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6).

[0458] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0459] (1) a polypeptide represented by general formula (XII):

[0460] aa12-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-a12-Pro-Phe

[0461] General formula (XII)

[0462] wherein a12 is selected from an amino acid with a positively charged side chain, an amino acid with a hydrophobic side chain, an amino acid with a polar uncharged side chain, or an amino acid with a negatively charged side chain, and aa12 is selected from C 11-25 hydrocarbyl-γGlu, carboxyl-C 10-24 hydrocarbyl-γGlu, C 11-25 hydrocarbyl-γGlu-mxOEG, carboxyl-C 10-24 hydrocarbyl-γGlu-mxOEG, C 11-25 hydrocarbyl-γGlu-nxPEG, carboxyl-C 10-24 hydrocarbyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or

[0463] (2) a polypeptide having at least 70% homology to the polypeptide represented by general formula (XII) and having the same function.

[0464] In certain embodiments, exemplary examples of a12 that can be used in the present disclosure include, but are not limited to, Arg, Lys, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gin, Asp, and Glu.

[0465] In certain embodiments, exemplary examples of aa12 that can be used in the present disclosure include, but are not limited to, C 11-25hydrocarbyl-γGlu, carboxyl-C 10-24 hydrocarbyl-γGlu, carboxyl-C 10-24 hydrocarbyl-γGlu-2xOEG, carboxyl-C 10-24 hydrocarbyl-γGlu-3xOEG or PEG.

[0466] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

[0467] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , or Y299 7.49 , thereby affecting G protein bias.

[0468] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide represented by general formula (XII).

[0469] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from:

[0470] C 19 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe (Disulfide bridge: 1-6);

[0471] C 17 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe (Disulfide bridge: 1-6);

[0472] COOHC 18 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe (Disulfide bridge: 1-6);

[0473] C 19 Alkyl-CO-2xγGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6);

[0474] C 19 Alkyl-CO-2xγGlu-DArg-DLeu-DArg-DAla-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6);

[0475] Ac-DLys(2xOEG-γGlu-C 19 Alkyl-CO)-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6);

[0476] DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6);

[0477] C 19 Alkyl-CO-γGlu-2xOEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6);

[0478] Ac-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 4-9);

[0479] Ac-Lys-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 4-9);

[0480] Ac-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 3-8);

[0481] Ac-Lys-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 3-8);

[0482] Ac-Arg-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 3-8);

[0483] Ac-Lys-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 5-10);

[0484] Ac-Leu-Arg-Gly-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 5-10);

[0485] Ac-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2 (Disulfide bridge: 4-9);

[0486] DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2 (Disulfide bridge: 4-9);

[0487] COOHC 16 alkyl-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2 (Disulfide bridge: 4-9);

[0488] COOHC 18 alkyl-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2 (Disulfide bridge: 4-9);

[0489] COOHC 16 alkyl-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2 (Disulfide bridge: 4-9);

[0490] COOHC 18 alkyl-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Leu-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2 (Disulfide bridge: 4-9);

[0491] COOHC16 Alkyl-CO-γGlu-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge: 4-9);

[0492] COOHC 18 Alkyl-CO-γGlu-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge: 4-9);

[0493] COOHC 18 Alkyl-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2(Disulfide bridge: 4-9);

[0494] COOHC 16 Alkyl-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2(Disulfide bridge: 4-9);

[0495] COOHC 18 Alkyl-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2(Disulfide bridge: 4-9);

[0496] COOHC 18 Alkyl-CO-γGlu-γGlu-DArg-DLeu-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 5-10);

[0497] COOHC 18Alkyl-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 5-10);

[0498] COOHC 18 Alkyl-CO-γGlu-γGlu-DArg-DLeu-DArg-DAla-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 6-11);

[0499] COOHC 18 Alkyl-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 6-11); and

[0500] COOHC 18 Alkyl-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Arg-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 7-12).

[0501] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0502] (1) a polypeptide represented by general formula (XIII):

[0503] aa13-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-a13-Phe

[0504] General formula (XIII)

[0505] wherein a13 is selected from an amino acid having a hydrophobic side chain, an amino acid having a polar uncharged side chain, an amino acid having a side chain with a negative charge, or His, and aa13 is selected from C 11-25 hydrocarbonyl-γGlu, carboxyl-C 10-24 hydrocarbonyl-γGlu, C11-25 Alkylacyl-γGlu-mxOEG, carboxyl-C 10-24 Alkylacyl-γGlu-mxOEG, C 11-25 Alkylacyl-γGlu-nxPEG, carboxyl-C 10-24 Alkylacyl-γGlu-nxPEG or PEG, and cysteine residues at positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5, n is an integer from 1 to 8; or

[0506] (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (XIII) and having the same function.

[0507] In certain embodiments, exemplary examples of aa13 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gin, Asp, Glu, and His.

[0508] In certain embodiments, exemplary examples of aa13 that can be used in the present disclosure include, but are not limited to, C 11-25 Alkylacyl-γGlu, carboxyl-C 10-24 Alkylacyl-γGlu, carboxyl-C 10-24 Alkylacyl-γGlu-2xOEG, carboxyl-C 10-24 Alkylacyl-γGlu-3xOEG and PEG.

[0509] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure have at least 70% homology with M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60, S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 in combination.

[0510] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 or Y299 7.49 in combination, thereby affecting G protein bias.

[0511] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide represented by general formula (XIII).

[0512] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from:

[0513] C 15 alkyl-CO-yGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Tyr-Phe (Disulfide bridge: 1-6);

[0514] C 17 alkyl-CO-yGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-DLeu-Phe (Disulfide bridge: 1-6);

[0515] COOHC 18 alkyl-CO-yGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-DLeu-Phe (Disulfide bridge: 1-6); and

[0516] COOHC 22 alkyl-CO-yGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-DLeu-Phe (Disulfide bridge: 1-6).

[0517] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0518] (1) a polypeptide represented by general formula (XIV):

[0519] aa14-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-a14

[0520] General formula (XIV)

[0521] wherein a14 is selected from the group consisting of an amino acid having a hydrophobic side chain, an amino acid having a polar uncharged side chain, an amino acid having a negatively charged side chain, His, or Nal, and aa14 is selected from the group consisting of C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5, and n is an integer from 1 to 8; or

[0522] (2) a polypeptide having at least 70% homology to the polypeptide represented by general formula (XIV) and having the same function.

[0523] In certain embodiments, exemplary examples of a14 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gin, Asp, Glu, and His.

[0524] In certain embodiments, exemplary examples of aa14 that can be used in the present disclosure include, but are not limited to, C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 10-24 alkanoyl-γGlu-2xOEG, carboxyl-C 10-24 alkanoyl-γGlu-3xOEG or PEG.

[0525] In certain embodiments, the Apelin-13 polypeptide mutant of the present disclosure is conjugated to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55Y264 6.51 K265 6.55 Y35 1.39 T295 7.39 Y299 7.49 F78 2.53 W85 2.60 Y88 2.63 Y93 ECL1 M113 3.36 R168 4.64 Y185 ECL2 E198 5.35 Y271 6.58 S275 6.62 M288 7.32 F291 7.35 P163 4.59 L201 5.38 V164 4.60 S205 5.42 P292 7.36 M288 7.32 Q180 ECL2 or Y182 ECL2 in combination.

[0526] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 F110 3.33 I109 3.32 K268 6.55 Y264 6.51 K265 6.55 Y35 1.39 T295 7.39 or Y299 7.49 in combination, thereby affecting G protein bias.

[0527] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide represented by the general formula (XIV).

[0528] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from:

[0529] C 19 alkyl-CO-yGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DTyr (Disulfide bridge: 1-6);

[0530] C 17Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro- DTyr (Disulfide bridge: 1-6); and

[0531] PEG5-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DTyr (Disulfide bridge: 1-6).

[0532] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0533] (1) a polypeptide represented by general formula (XV):

[0534] aa15-Cys-a15-b15-c15-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe

[0535] General formula (XV)

[0536] wherein a15 and b15 are each independently selected from a positively charged side chain amino acid or Pro, c15 is selected from a hydrophobic side chain amino acid or a positively charged side chain amino acid, aa15 is selected from C 11-25 Alkanoyl-γGlu, Carboxy-C 10-24 Alkanoyl-γGlu, Carboxy-C 10-24 Alkanoyl-γGlu-2xOEG, Carboxy-C 10-24 Alkanoyl-γGlu-3xOEG or PEG, and the cysteine residues at positions 1 and 5 form a disulfide bond; or

[0537] (2) a polypeptide having at least 70% homology to the polypeptide represented by general formula (XV) and having the same function.

[0538] In certain embodiments, exemplary examples of a15 that can be used in the present disclosure include, but are not limited to, Arg, Lys, and Pro.

[0539] In certain embodiments, exemplary examples of b15 that can be used in the present disclosure include, but are not limited to, Arg, Lys, and Pro.

[0540] In certain embodiments, exemplary examples of c15 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Arg, and Lys.

[0541] In certain embodiments, exemplary examples of aa15 that can be used in the present disclosure include, but are not limited to, C 11-25 hydrocarbon CO- yGlu, COOHC 10-24 hydrocarbon CO- yGlu, COOHC 10-24 hydrocarbon CO- yGlu-2xOEG, COOHC 10-24 hydrocarbon CO- yGlu-3xOEG or PEG.

[0542] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

[0543] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide represented by general formula (XV).

[0544] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from:

[0545] C 19 alkyl-CO- yGlu-Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0546] C17 alkyl-CO-γGlu-2xOEG-Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and

[0547] COOHC 22 alkyl-CO-γGlu-2xOEG-Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

[0548] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0549] (1) a polypeptide represented by general formula (XVI):

[0550] aa16-Cys-a16-b16-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe

[0551] General formula (XVI)

[0552] wherein a16 is selected from an amino acid with a positively charged side chain or Pro, b16 is selected from an amino acid with a hydrophobic side chain, an amino acid with a positively charged side chain, or Pro, and aa16 is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu-2xOEG, carboxy-C 10-24 alkanoyl-γGlu-3xOEG or PEG, and the cysteine residues at positions 1 and 4 form a disulfide bond; or

[0553] (2) a polypeptide having at least 70% homology to the polypeptide represented by general formula (XVI) and having the same function.

[0554] In certain embodiments, exemplary examples of a16 that can be used in the present disclosure include, but are not limited to, Arg, Lys, and Pro.

[0555] In certain embodiments, exemplary examples of b16 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Arg, Lys, and Pro.

[0556] In certain embodiments, exemplary examples of aa16 that can be used in the present disclosure include, but are not limited to, C 11-25 alkanoyl-γGlu, carboxy-C10-24 Alkyl-CO-γGlu-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); 10-24 Alkyl-CO-γGlu-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); 10-24 Alkyl-CO-γGlu-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0557] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

[0558] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide represented by the general formula (XVI).

[0559] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from:

[0560] C 19 Alkyl-CO-γGlu-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0561] COOHC 22Alkyl-CO-γGlu-2xOEG-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and

[0562] PEG5-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

[0563] In yet another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0564] (1) a polypeptide represented by general formula (XVII):

[0565] *-Arg-a17-b17-Leu-*-His-c17-Gly-Pro-DMet-Gly-Pro-Phe

[0566] General formula (XVII)

[0567] wherein a17and b17are each independently selected from an amino acid with a positively charged side chain, or a17and / or b17are absent, c17is an amino acid with a positively charged side chain, * is a non-natural amino acid, and a loop structure is formed between *; or

[0568] (2) a polypeptide having at least 70% homology to the polypeptide represented by general formula (XVII) and having the same function.

[0569] In certain embodiments, exemplary examples of a17and b17that can be used in the present disclosure include, but are not limited to, Arg, Lys, and Pro.

[0570] In certain embodiments, a17and b17that can be used in the present disclosure are both absent.

[0571] In certain embodiments, exemplary examples of b16that can be used in the present disclosure include, but are not limited to, Arg and Lys.

[0572] In certain embodiments, exemplary examples of * that can be used in the present disclosure include, but are not limited to:

[0573] In certain embodiments, exemplary examples of a loop structure formed between * that can be used in the present disclosure include, but are not limited to, a staple structure.

[0574] In certain embodiments, exemplary examples of a loop structure formed between * that can be used in the present disclosure include, but are not limited to In certain embodiments, exemplary examples of a loop structure formed between * that can be used in the present disclosure include, but are not limited to

[0575] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 F110 3.33 I109 3.32 K268 6.55 Y264 6.51 K265 6.55 Y35 1.39 T295 7.39 Y299 7.49 F78 2.53 W85 2.60 Y88 2.63 Y93 ECL1 M113 3.36 R168 4.64 Y185 ECL2 E198 5.35 Y271 6.58 S275 6.62 M288 7.32 F291 7.35 P163 4.59 L201 5.38 V164 4.60 S205 5.42 P292 7.36 M288 7.32 Q180 ECL2 or Y182 ECL2 .

[0576] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide represented by the general formula (XVII).

[0577] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from:

[0578] In yet another aspect, the present disclosure relates to Apelin-13 polypeptide mutants comprising

[0579] (1) a polypeptide represented by the general formula (XVIII):

[0580] a18-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-b18-Pro-Phe

[0581] General formula (XVIII)

[0582] wherein a18 is selected from:

[0583] b18 is selected from an amino acid having a hydrophobic side chain or an amino acid having a negatively charged side chain, and the cysteine residues at positions 1 and 6 form a disulfide bond; or

[0584] (2) a polypeptide having at least 70% homology to a polypeptide represented by general formula (XVIII) and having the same function.

[0585] In certain embodiments, exemplary examples of b18 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Asp, and Glu.

[0586] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

[0587] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , or Y2997.49 bind, thereby influencing G protein bias.

[0588] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide represented by general formula (XVIII).

[0589] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from the group consisting of:

[0590] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.

[0591] In another aspect, the present disclosure relates to Apelin-13 polypeptide mutants comprising

[0592] (1) a polypeptide represented by general formula (XIX):

[0593] a19-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-b19-c19-Pro-Phe

[0594] General formula (XIX)

[0595] wherein a19 is selected from the group consisting of:

[0596] b19 is selected from the group consisting of an amino acid having a hydrophobic side chain or an amino acid with a negatively charged side chain, c19 is selected as an amino acid having a hydrophobic side chain, and the cysteine residues at positions 1 and 6 form a disulfide bond; or

[0597] (2) a polypeptide having at least 70% homology to the polypeptide represented by general formula (XIX) and having the same function.

[0598] In certain embodiments, exemplary examples of b19 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

[0599] In certain embodiments, exemplary examples of c19 that can be used in the present disclosure include, but are not limited to, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Asp, and Glu.

[0600] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51, K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 in combination.

[0601] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure bind to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 or Y299 7.49 in combination, thereby affecting G protein bias.

[0602] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure consist of a polypeptide represented by the general formula (XIX).

[0603] In certain embodiments, the Apelin-13 polypeptide mutants of the present disclosure are selected from the group consisting of:

[0604] SEQ ID NO: 2 and SEQ ID NO: 5.

[0605] In yet another aspect, the present disclosure relates to a pharmaceutical composition comprising an Apelin-13 polypeptide mutant as described herein, and a pharmaceutically acceptable carrier, diluent or excipient.

[0606] In another aspect, the present disclosure relates to a method of treating or preventing a disease or condition responsive to agonism of the APLNR receptor, comprising administering to an individual in need of said method a therapeutically or prophylactically effective amount of an Apelin-13 polypeptide mutant of the present disclosure or a pharmaceutical composition of the present disclosure.

[0607] In certain embodiments, exemplary examples of diseases or conditions that can be used in the present disclosure include, but are not limited to, maintenance of muscle mass, fat loss, acute decompensated heart failure, acute heart failure, chronic heart failure, pulmonary hypertension, atrial fibrillation, kidney disease, muscle building, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular disease, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes, obesity, peripheral arterial disease, cerebrovascular accident, transient ischemic attack, traumatic brain injury, amyotrophic lateral sclerosis, burn, preeclampsia, anti-aging, analgesia, sleep apnea syndrome, pulmonary embolism, asthma, stress-induced gastric ulcer, liver fibrosis, lung injury, brain injury, ischemic stroke, cancer, Alzheimer's disease, Parkinson's disease, stroke, renal hypertension, and neurodegenerative disease.

[0608] In certain embodiments, exemplary examples of individuals that can be used in the present disclosure include, but are not limited to, mammals.

[0609] In certain embodiments, exemplary examples of individuals that can be used in the present disclosure include, but are not limited to, humans.

[0610] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant of the present disclosure for use in the treatment or prevention of a disease or condition responsive to agonism of the APLNR receptor.

[0611] In yet another aspect, the present disclosure relates to use of an Apelin-13 polypeptide mutant of the present disclosure in the manufacture of a medicament for the treatment or prevention of a disease or condition responsive to agonism of the APLNR receptor.

[0612] Hereinafter, the present disclosure will be explained in detail by the following examples in order to better understand various aspects of the present application and its advantages. It is to be understood that the following examples are non-limiting and are merely illustrative of certain embodiments of the present disclosure.

[0613] The reagents and apparatus used in the examples of the present disclosure are all conventional and commercially available.

[0614] General preparation process of polypeptides

[0615] Polypeptide sequence: if sequence is ARSS

[0616] I. Synthesis of polypeptides

[0617] Synthetic sequence: synthesis from C-terminal to N-terminal of the peptide chain

[0618] a. Swelling of the resin: weigh the calculated amount of fmoc-Ser(tbu)-OH resin into the reaction column, soak for 30 minutes with DMF, and dry.

[0619] b. Deprotection:

[0620] Add an appropriate amount of deprotection solution (piperidine = 20% hexahydro-pyridine + 80% DMF) to the reaction column, stir and agitate for 30 minutes under nitrogen, dry, add an appropriate amount of DMF to the reaction column, agitate for 2 minutes under nitrogen, dry, and repeat the operation 6 times.

[0621] c. Weighing:

[0622] Weigh 3 times the molar amount of the protected amino acid, and then weigh 2.85 times the molar amount of HBTU (condensing agent) for standby

[0623] d. Feeding:

[0624] Add the prepared protected amino acid and HBTU to the reaction column, and then add 6 times the molar amount of NMM (organic base) to the resin, stir and agitate for 30 minutes under nitrogen.

[0625] e. Post-reaction washing:

[0626] Dry the solution in the reaction column, wash with an appropriate amount of DMF, agitate for 2 minutes under nitrogen, dry, and repeat the operation 3 times.

[0627] f. Detection:

[0628] Take an appropriate amount (10-20) of resin in a small test tube, add two drops of A, B, and C liquid respectively. Put it into a dry heater and heat for 3 minutes (110 degrees Celsius). After taking it out, if the solution is blue and the resin has a colored and opaque color, the reaction is not complete and needs to be re-reacted again; if the solution is slightly yellow and the resin is colorless and transparent, the reaction is complete and the next amino acid can be connected. Repeat the above b-f five steps until the last amino acid is connected. That is, ADSS is obtained. Finally, remove the Fmoc protection group on FmocAla-OH to expose the N-terminal. The last condensed amino acid is octadecanedioic acid mono-t-butyl ester. This amino acid does not have a Fmoc group protection, so it does not need to be deprotected after condensation.

[0629] g. Washing and drying after synthesis is complete:

[0630] After the last amino acid is coupled and the resin is washed, the resin is drained, and the appropriate amount of methanol is added to the column. The column is flushed with nitrogen for 2 minutes, drained, and the appropriate amount of DCM is added to the column. The column is flushed with nitrogen for 2 minutes, drained, and the procedure is repeated three times. The final rinse is performed with methanol, and the column is flushed with nitrogen for 2 minutes, drained, and the procedure is repeated two times. The resin is then transferred to a suitable container and dried in a vacuum desiccator for 12 hours.

[0631] II. Cleavage of the polypeptide

[0632] Cleavage: The dried resin is transferred to a suitable round bottom flask, and the appropriate amount of cleavage solution (1 g / 10 ml) is added. The flask is placed in a constant temperature shaker and shaken at 25°C for 2 hours.

[0633] Filtration: The resin particles are filtered out using a 50 mL fritted funnel, and the filtrate is poured into a glass container in a centrifuge tube. Anhydrous ether is added to the container in an amount of 6-8 times the volume of the filtrate, and the mixture is stirred while the white solid precipitates. The white solid is the desired crude polypeptide.

[0634] Washing: The white solid is transferred to a centrifuge tube, which is sealed and centrifuged at 4000 rpm for 3 minutes. The supernatant is discarded, and anhydrous ether is added to the tube. The mixture is stirred with a glass rod, and the tube is centrifuged again. The procedure is repeated 5 times.

[0635] Liquid-phase ring-closing synthesis:

[0636] The crude linear peptide is diluted with DMF to a concentration of 10 -3 mol / L and placed in an ice bath to dissolve completely. An amount of 1.5 times the volume of HOAt, 1.5 times the volume of HATU, and 10 times the volume of DIEA are added, and the mixture is stirred in the ice bath for 2 hours. The ice bath is removed, and the mixture is stirred at room temperature. The solid residue is obtained after distillation under reduced pressure.

[0637] Drying: The polypeptide after 5 washes is placed in a vacuum desiccator and dried for 24 hours. The final white powder is the desired crude polypeptide, which is weighed and purified.

[0638] Note: Amino acids and reagents used

[0639] A. Amino acids

[0640] Fmoc-Arg(pbf)-OH

[0641] Fmoc-Ala-OH

[0642] Fmoc-Ser(tbu)-OH

[0643] B. Condensing agents and organic bases

[0644] HBTU

[0645] NMM

[0646] C. Solvent and nitrogen:

[0647] DMF

[0648] DCM

[0649] Methanol

[0650] Hexahydropyridine

[0651] 98% N2

[0652] D. Detection solution

[0653] A solution: 80% phenol + 20% anhydrous ethanol

[0654] B solution: redistilled pyridine

[0655] C solution: 5 g of indantrione + 100 mL of anhydrous ethanol

[0656] E. Preparation of deprotection solution

[0657] 20% hexahydropyridine + 80% DMF

[0658] G. Cleavage solution for cutting

[0659] 87.5% TFA + 5% benzyl mercaptan + 2.5% phenol + 2.5% EDT + 2.5% H2O

[0660] III. Purification method steps

[0661] 1. First, perform a 10-100% analysis of the crude product to determine the retention time of the main peak, then perform a linear gradient analysis.

[0662] 2. The sample is dissolved in water and acetonitrile (4:1) to fully dissolve the polypeptide.

[0663] 3. Take 200 mg of polypeptide and dissolve it in a 10 mL dissolution bottle. After ultrasonic dissolution, filter the clear liquid through a 0.45 μm organic phase filter.

[0664] 4. Perform a linear gradient preparation to collect the peak and determine the correctness of the MS.

[0665] 5. Track the purity of the collected liquid and feedback the amount of liquid that meets the purity requirements.

[0666] 6. Perform low-temperature concentration on the collected qualified liquid, with a concentration temperature of 38°C, and concentrate to a liquid amount of 50-100 mL (in special cases, generally not concentrated directly frozen).

[0667] 7. Place the concentrated liquid amount in a 100 mL beaker and freeze it into a solid state using liquid nitrogen or dry ice.

[0668] 8. Drying

[0669] 9. Weighing

[0670] General characterization method of polypeptides

[0671] I. Ability to determine whether the peak of interest is the prepared polypeptide sample by MS during purification:

[0672] 1. Prepare the sample to be tested, ensuring that the sample is suitable for mass spectrometry analysis, usually requiring dissolution and extraction.

[0673] 2. Adjust instrument parameters

[0674] Select ion source: select appropriate ion source according to sample properties, such as electrospray ion source (ESI) or chemical ion source (CI) coupled with gas chromatography-mass spectrometry.

[0675] 3. Set the mass spectrometry analysis mode: select the ion monitoring mode for mass spectrometry analysis.

[0676] 4. Perform mass spectrometry analysis

[0677] Start the ion source and wait for it to stabilize, inject the polypeptide sample into the mass spectrometer.

[0678] 5. Data acquisition

[0679] Start mass spectrometry analysis and collect mass spectrometry data in real time, including mass spectrum and relative abundance.

[0680] 6. Analyze mass spectrometry data

[0681] Use mass spectrometry software to analyze the mass spectrum and identify proton peaks or molecular ion peaks. Determine whether the target polypeptide is present in the sample based on the mass spectrometry data.

[0682] II. Subsequently, detect the purity of the polypeptide by HPLC:

[0683] 1. Turn on the HPLC workstation, condense the flow phase pipeline, and connect the detection system.

[0684] 2. Adjust the flow rate, try the pressure first when using new mobile phase, the greater the flow rate, the greater the pressure, generally not more than 2000. Click on injure, select the appropriate flow rate, click on, baseline, and observe the baseline.

[0685] 3. Set the sample method, stabilize the flow for 2-5 min, baseline zero, loading-inject conversion, and set the sample time.

[0686] The obtained polypeptide sample is filtered and loaded. After the method is completed, click postrun to record data and make marks, etc. After all samples are completed, use the above method to run a baseline and wash away the remaining substances.

[0687] 4. The proportion of the main peak to all peaks is the purity of the polypeptide sample.

[0688] The general characterization method of the fusion protein uses SDS-PAGE electrophoresis to detect whether it is the target protein sequence and determine the protein purity.

[0689] General preparation method of fusion protein

[0690] The target gene is constructed on a TAC expression vector, transformed into E. coli for amplification and preservation of bacterial liquid, and used for subsequent expression and purification in E. coli.

[0691] 1. Inoculation: 1 tube of glycerol bacteria + 25 ml of LB (1:1000 A+), 37°C shaking overnight, 5 mL of bacterial liquid: 500 mL of LB (1:1000 A+) to a large bottle (LB in a large bottle is sterilized in advance), 37°C shaking for 2-3 h, OD value is measured every hour, when the OD value is 0.6-0.8, the bacteria are placed at 16°C, 220 rpm shaking.

[0692] 2. Induction: 0.5M IPTG final concentration 0.3mM added to the bacterial liquid, 16°C, 220 rpm shaking for 20h.

[0693] 3. Collect bacteria: 6000g, 10min.

[0694] 4. Resuspend: 1:5-10 add lysis buffer to resuspend the bacterial body, then pour into a beaker. Add lysozyme (final concentration 0.5mg / mL), DNase (1:100) and 1mM PMSF.

[0695] 5. Stir: 4°C stirring for 0.5h.

[0696] 6. Lysis: use high-pressure homogenizer to break the cells to clarify the solution.

[0697] 7. Centrifugation: 30000g, 4°C, 30min.

[0698] 8. Collect supernatant: collect the centrifuged supernatant into a beaker, and add 30mM imidazole to the supernatant.

[0699] 9. Equilibrate Ni column: take 3mL beads to the column, first water and then equilibrate buffer, wash 5CV respectively.

[0700] 10. Column hanging: slowly add the supernatant to the Ni column. Repeat 1 to 2 times.

[0701] 11. Wash: Wash buffer wash for about 20 CV.

[0702] 12. Elute: Elution buffer elute protein, collect eluate.

[0703] 13. Measure concentration: Detect protein content.

[0704] 14. Concentrate protein eluate: Concentrate to 500 μL with 10 KD concentrator tube.

[0705] 15. Centrifuge: After concentration, transfer to 1.5 mL EP tube and centrifuge at 12000 rpm for 10 min.

[0706] 16. Pass through molecular sieve.

[0707] 17. Concentrate: Collect molecular sieve out peak position protein eluate, concentrate to 250 μL.

[0708] 18. Centrifuge, transfer to new EP tube, measure concentration, aliquot to 0.2 mg per tube, quick freeze in liquid nitrogen, store at -80 °C.

[0709] General characterization method of fusion protein

[0710] Use SDS-PAGE electrophoresis to detect whether it is the target protein sequence and determine the purity of the protein.

[0711] The steps are as follows:

[0712] 1. Prepare the gel:

[0713] Prepare the separation gel and the concentration gel.

[0714] Mix an appropriate amount of acrylamide and bisacrylamide with SDS, TEMED (tetramethyl ethylenediamine) and APS (ammonium persulfate) to prepare the separation gel solution.

[0715] Add the separation gel solution between the glass plates and insert the comb to form sample wells.

[0716] After waiting for the separation gel to solidify, add the concentration gel solution on top and insert the comb to form sample wells.

[0717] 2. Sample preparation:

[0718] Mix the protein sample with the SDS sample buffer and heat to 95 °C to denature the protein.

[0719] Add a reducing agent such as DTT (dithiothreitol) or β-mercaptoethanol to break the disulfide bonds in the protein.

[0720] Loading:

[0721] Place the solidified gel and glass plates in the electrophoresis tank and add the electrophoresis buffer.

[0722] Carefully remove the comb to expose the sample wells.

[0723] Add denatured protein sample to the sample wells.

[0724] 3. Electrophoresis:

[0725] Turn on the power supply and start the electrophoresis. Typically, the starting voltage is set at 80-100 volts, and after the sample has entered the resolving gel, the voltage can be increased to around 200 volts.

[0726] During the electrophoresis, proteins are separated in the gel according to their molecular weights.

[0727] 4. Staining and destaining:

[0728] After the electrophoresis is complete, remove the gel and stain the proteins, typically using Coomassie Brilliant Blue R-250 or silver staining.

[0729] After staining, remove the unbound dye using a destaining solution (such as methanol / acetic acid) to make the protein bands clearly visible.

[0730] 5. Analysis:

[0731] The gel can be photographed using an imaging system, or analyzed using a gel scanner.

[0732] Protein bands can be quantitatively analyzed using appropriate software, such as molecular weight estimation, protein content determination, etc.

[0733] Preparation Example 1

[0734] COOHC 16 alkyl

[0735] Preparation of -CO-γGlu-Arg-Lys-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Dleu-Pro-Phe (Disulfide bridge: 5-10)

[0736] The synthetic raw materials are: lysine, arginine, proline, leucine, glutamic acid, histidine, glycine, D-methionine, D-leucine, alanine, C18 diacid, gamma glutamic acid; the reagents used include: condensing agent and organic base HBTU and NMM, DMF, DCM, methanol, piperidine, 98% N2; detection solution (A solution: 80% phenol + 20% anhydrous ethanol; B solution: redistilled pyridine; C solution: 5g indantrione + 100mL anhydrous ethanol); deprotection solution: 20% piperidine + 80% DMF; cleavage solution for cutting: 87.5% TFA + 5% benzyl mercaptan + 2.5% phenol + 2.5% EDT + 2.5% H2O.

[0737] The synthesis steps are as follows:

[0738] a. Resin swelling: weigh 1g of fmoc-Phe(tbu)-OH resin into the reaction column, add 15mL DMF and soak for 30 minutes, and then dry (the first amino acid Phe is connected to the resin).

[0739] b. Deprotection: add 25ml deprotection solution (piperidine = 20% piperidine + 80% DMF) to the reaction column, stir and agitate for 30 minutes under nitrogen, dry, add 25mL DMF to the reaction column, agitate for 2 minutes under nitrogen, dry, and repeat the operation 6 times.

[0740] c. Weighing: weigh 4.8g of protected amino acid, and then weigh 0.562g of HBTU (condensing agent) for standby.

[0741] d. Feeding: add the prepared protected amino acid and HBTU to the reaction column, and then add 0.608mL of NMM (organic base) to the resin, and agitate for 30 minutes under nitrogen.

[0742] e. Post-reaction washing: dry the solution in the reaction column, add 25mL DMF for washing, agitate for 2 minutes under nitrogen, dry, and repeat the operation 3 times.

[0743] f. Detection: take 20 resin samples in small test tubes, and add two drops of A, B, and C solution respectively. Put them into a dry heater and heat for 3 minutes (110 degrees Celsius).

[0744] After taking out, if the solution shows blue color and the resin has a colored and opaque color, the reaction is not complete and needs to be re-reacted again; if the solution color is slightly yellow and the resin is colorless and transparent, the reaction is complete, and the next amino acid Pro is connected, and then the above five steps b-f are repeated to connect Phe, Pro, Dleu, DMet, Pro, Gly, Lys, His, Cys, Glu, Leu, Arg, Pro, Arg, Cys, Arg, Lys, and Arg respectively.

[0745] The title sequence is obtained. The last protecting group Fmoc on FmocAla-OH is removed to expose the N-terminus. The last condensed amino acid is octadecanedioic acid mono-tert-butyl ester and gamma glutamic acid, which has no Fmoc group protection, so no Fmoc protection is needed after condensation.

[0746] g. Washing and drying after the completion of synthesis:

[0747] After the last amino acid is connected and the washing after deprotection is completed, the reaction column is drained, an appropriate amount of methanol is added, and nitrogen is blown for 2 minutes. The reaction column is drained, and an appropriate amount of DCM is added, and nitrogen is blown for 2 minutes. The reaction column is drained, and the operation is repeated 3 times. Finally, an appropriate amount of methanol is added to the reaction kettle, and nitrogen is blown for 2 minutes. The reaction kettle is drained, and the operation is repeated 2 times. The resin is loaded into a suitable container and placed in a vacuum dryer for 12 hours before cleavage

[0748] II. Cleavage of the polypeptide

[0749] Cleavage: The dried resin is loaded into a suitable round-bottom flask, an appropriate amount of prepared cleavage solution (1 g / 10 mL) is added, and the flask is placed in a constant-temperature shaker at 25°C for constant-temperature shaking for 2 hours.

[0750] Filtration: The resin particles are filtered out using a 50 mL sand core funnel, and then the filtrate is poured into a glass container of a centrifuge tube. Anhydrous ether is added in an amount of 6-8 times the volume, and the white solid that separates out is the desired crude polypeptide.

[0751] Washing: The white solid is divided into centrifuge tubes, which are sealed and placed in a centrifuge at a speed of 4000 r / min for 3 minutes. The upper clear liquid is discarded, and ether is added. The mixture is stirred with a glass rod and centrifuged again. This operation is repeated 5 times.

[0752] Liquid-phase ring synthesis:

[0753] The crude linear peptide is diluted with DMF to 10 -3 mol / L, and placed in an ice bath to dissolve completely. An amount of HOAt 1.5 times that of the peptide, an amount of HATU 1.5 times that of the peptide, and an amount of DIEA 10 times that of the peptide are added. The mixture is stirred in the ice bath for 2 hours, and then removed from the ice bath and stirred at room temperature. The solid residue is obtained after distillation under reduced pressure.

[0754] Drying: The polypeptide after 5 times of washing is placed in a vacuum dryer and dried for 24 hours.

[0755] The final white powder obtained is the crude polypeptide, which is weighed and purified.

[0756] III. Purification method steps

[0757] 1. First, analyze 10-100% of the crude product to determine the retention time of the main peak, then perform a linear gradient analysis.

[0758] 2. The sample can be dissolved in water and acetonitrile (4:1) to fully dissolve the polypeptide.

[0759] 3. Dissolve 200 mg of the polypeptide in 10 ml of a dissolution bottle, and after ultrasonic dissolution, filter the clear solution with a 0.45 um organic phase filter.

[0760] 4. Prepare the peak collection with a linear gradient, and determine the correctness of the MS.

[0761] 5. Track the purity feedback of the collected liquid, and obtain the liquid amount that meets the purity requirements.

[0762] 6. Low-temperature concentration is performed on the qualified liquid collected, the concentration temperature is 38°C, and the liquid amount is concentrated to 50-100 mL (in special cases, generally not concentrated directly frozen)

[0763] 7. Place the concentrated liquid amount in a 100 mL beaker, and freeze it into a solid state using liquid nitrogen or dry ice.

[0764] 8. Drying

[0765] MS characterization: Mass spectrum shows the mass-to-charge ratio of the polypeptide, three target peaks: [M+5H] 5+ = 507.1, [M+4H] 4+ = 633.6, [M+3H] 3+ = 844.4. The target peak is detected at these positions, so the purified polypeptide is indeed the title polypeptide sequence.

[0766] HPLC results show that the purity of the title polypeptide is 95.61%.

[0767] Preparation Example 2

[0768] C 19 alkyl

[0769] Preparation of -CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 2-7)

[0770] The preparation process is described in Preparation Example 1, and the required amino acid raw materials are cysteine, alanine, proline, arginine, leucine, cysteine, histidine, lysine, glycine, D-methionine, phenylalanine, monotert-butyl octadecanedioate, and gamma glutamic acid. After the first amino acid reaction is complete, the next amino acid Pro is connected, and then the above steps b-f are repeated to connect Gly, DMet, Pro, Gly, Lys, His, Cys, Leu, Arg, Pro, Arg, and Cys, respectively. The title sequence is obtained. Finally, the protecting group Fmoc on FmocPhe-OH is removed to expose the N-terminus. The last condensed amino acid is monotert-butyl octadecanedioate and gamma glutamic acid, which do not have a Fmoc group protection, so no Fmoc protection is needed after condensation.

[0771] MS characterization: mass spectrum shows the mass-to-charge ratio of the polypeptide, three target peaks: [M+4H] 4+ = 513.0, [M+3H] 3+ = 683.6, [M+2H] 2+ = 1025.6. The target peak is detected at these three positions, so the purified polypeptide sequence is indeed the title polypeptide sequence.

[0772] HPLC results show that the purity of the title polypeptide is 95.17%.

[0773] The amino acid sequence of the wild-type apelin-13 polypeptide is Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe (represented by single letters as QRPRLSHKGPMPF).

[0774] The MM07 amino acid sequence is Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Met-Pro-Phe (disulfide bridge 1-6, i.e., the Cys at positions 1 and 6 form a disulfide bond) (represented by single letters as CRPRLCHKGPMPF (disulfide bridge 1-6)).

[0775] Preparation Example 3

[0776] Preparation of FC fusion protein:

[0777] The sequence of the target protein synthesized is as follows:

[0778] The target gene is constructed on a TAC expression vector, transformed into E. coli for amplification and preservation of bacterial liquid, and used for subsequent expression and purification in E. coli.

[0779] 1. Inoculation: 1 tube of glycerol bacteria + 25ml LB (1:1000 A+), 37°C shaker overnight, 5mL bacteria: 500mL LB (1:1000 A+) transfer to a large bottle (LB in the large bottle is sterilized in advance), 37°C shaker for 2-3h, OD value is measured every hour, when the OD value is 0.6-0.8, the bacteria is placed at 16°C, 220rpm shaker for pre-cooling.

[0780] 2. Induction: 0.5M IPTG final concentration 0.3mM is added to the bacteria, 16°C, 220rpm shaker for 20h.

[0781] 3. Collect bacteria: 6000g, 10min

[0782] 4. Resuspend: 1:5-10 add lysis buffer to resuspend the bacteria, then pour into a beaker. Add lysozyme (final concentration 0.5mg / mL), DNase (1:100) and 1mM PMSF.

[0783] 5. Stir: 4°C stirring for 0.5h.

[0784] 6. Lysis: the cells are broken by using a high-pressure homogenizer until the solution is clear.

[0785] 7. Centrifugation: 30000g, 4°C, 30min.

[0786] 8. Collect supernatant: the centrifuged supernatant is collected in a beaker, and 30mM imidazole is added to the supernatant.

[0787] 9. Equilibrate Ni column: take 3ml beads to the column, first water and then equilibrate buffer, respectively wash 5CV.

[0788] 10. Column hanging: slowly add the supernatant to the Ni column. Repeat 1 to 2 times.

[0789] 11. Wash: wash with about 20CV of washing buffer.

[0790] 12. Elution: elute the protein with elution buffer, and collect the eluate.

[0791] 13. Measure concentration: detect the protein content.

[0792] 14. Concentrate protein eluate: concentrate to 500μL with 10KD concentration tube.

[0793] 15. Centrifugation: after concentration, transfer to a 1.5mL EP tube and centrifuge at 12000rpm for 10min.

[0794] 16. Pass through molecular sieve.

[0795] 17. Concentrate: collect the eluate of the target protein at the peak position of the molecular sieve, and concentrate to 250μL.

[0796] 18. Centrifuge, transfer to new EP tubes, measure concentration, aliquot to 0.2 mg per tube, snap frozen in liquid nitrogen, store at -80 °C.

[0797] 19. SDS-page characterization: the purified target protein was subjected to protein electrophoresis, and the protein maker was compared to determine that the protein was indeed in the position of the target protein, indicating that the purified protein was indeed the target protein sequence. In addition, the banding determined that the purity of the target protein was high, which could reach more than 90%. The results are shown in Figure 3.

[0798] The remaining mutant polypeptide sequences used in the present application are shown in Table 1.

[0799] Table 1

[0800] Preparation Example 4

[0801] C 19 alkyl

[0802] Preparation of -CO-2xγGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6)

[0803] Preparation process is described in Preparation Example 1, the required amino acid raw material is cysteine, alanine, proline, arginine, leucine, cysteine, histidine, lysine, glycine, D-methionine, D-leucine, D-arginine, phenylalanine, nonadecyl carboxylic acid / halide, gamma glutamic acid. Other method Preparation Example 1, take the resin with amide linker, after cleavage, automatically form C-terminal amide, load the resin into SPPS reaction column. Sufficiently swell the resin with DMF (dimethylformamide) for at least 30 minutes. Treat the resin with 20% piperidine / DMF solution for 2 times, 5-10 minutes each time, to remove the initial Fmoc protecting group on the resin. Mix Fmoc-Phe(tBu)-OH (Fmoc protected phenylalanine, tBu protected carboxyl) with DIC (diisopropyl carbodiimide) and Oxyma Pure (or HOBt), after activation, add to the resin. The reaction is usually carried out in DMF, stir for 30-60 minutes. After the first amino acid reaction is complete, start to link the next amino acid Pro, then repeat the above five steps b-f, respectively, to link DLeu, DMet, Pro, Gly, Arg, His, Cys, Leu, Arg, Pro, Arg, Cys, DArg, Dleu, DArg. Then remove the protection group Fmoc on FmocPhe-OH, expose the N-terminal. The last condensed amino acid is C 19 alkyl carboxylic acid and gamma glutamic acid, these amino acids do not have Fmoc group protection, so after condensation, there is no need to remove Fmoc protection. Subsequent cleavage purification steps are shown in Preparation Example 1.

[0804] MS characterization: mass spectrum shows the mass-to-charge ratio of the polypeptide, 5 target peaks: [M+6H] 6+ = 449.1, [M+5H] 5+ = 538.7, [M+4H] 4+ = 673.0, [M+3H] 3+ = 897.0, [M+2H] 2+ = 1345.0. The target peak is detected at the 5 positions, therefore, the purified is indeed the title polypeptide sequence.

[0805] HPLC results show that the purity of the title polypeptide is 96.20%.

[0806] Preparation Example 5

[0807] C 19 alkyl

[0808] Preparation of -CO-2xγGlu-DArg-DLeu-DArg-DAla-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 1-6)

[0809] The preparation process is as described in Preparation Example 1, and the required amino acid raw materials are cysteine, alanine, proline, arginine, leucine, cysteine, histidine, lysine, glycine, D-methionine, D-leucine, D-arginine, D-alanine, phenylalanine, nonadecyl carboxylic acid / halide, and γ-glutamic acid. Other methods are as described in Preparation Example 1, and the resin with an amide linker is loaded into an SPPS reaction column after cleavage to automatically form a C-terminal amide. The resin is fully swollen in DMF (dimethylformamide) for at least 30 minutes. The resin is treated with a 20% piperidine / DMF solution twice for 5-10 minutes each time to remove the initial Fmoc protecting group on the resin. Fmoc-Phe(tBu)-OH (Fmoc-protected phenylalanine, tBu-protected carboxyl) is mixed with DIC (diisopropyl carbodiimide) and Oxyma Pure (or HOBt) and added to the resin after activation. The reaction is usually carried out in DMF, and stirring is performed for 30-60 minutes. After the first amino acid reaction is complete, the next amino acid Pro is linked, and then the above steps b-f are repeated to link DLeu, DMet, Pro, Gly, Arg, His, Cys, Leu, Arg, Pro, Arg, Cys, DArg, DAla, DArg, DArg, Dleu, DArg, respectively. Then the protecting group Fmoc on FmocPhe-OH is removed to expose the N-terminus. The last condensed amino acid is C 19 alkyl carboxylic acid and γ-glutamic acid, which do not have Fmoc group protection, so no Fmoc removal is required after condensation. The subsequent cleavage and purification steps are as shown in Preparation Example 1.

[0810] MS characterization: Mass spectrometry shows the mass-to-charge ratio of the polypeptide, 4 target peaks: [M+6H] 6+ = 498.7, [M+5H] 5+ = 598.3, [M+4H] 4+ = 747.6, [M+3H] 3+ = 996.4. The target peaks are detected at all 4 positions, so the purified product is indeed the title polypeptide sequence.

[0811] HPLC results show that the purity of the title polypeptide is 96.30%.

[0812] Preparation Example 6

[0813] Ac-DLys(2xOEG-yGlu-C 19 alkyl

[0814] Preparation of Ac-DLys(2xOEG-yGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 1-6)

[0815] Preparation process is described in Preparation Example 1, the required amino acid raw materials are cysteine, alanine, proline, arginine, leucine, cysteine, histidine, lysine, glycine, D-methionine, D-leucine, D-arginine, phenylalanine, nonadecyl carboxylic acid / halide, gamma glutamic acid, oligoethylene glycol. Other methods are described in Preparation Example 1, the resin with amide linker is taken, and the C-terminal amide is automatically formed after cleavage. The resin is loaded into the SPPS reaction column. The resin is fully swollen with DMF (dimethylformamide) for at least 30 minutes. The resin is treated with 20% piperidine / DMF solution for 2 times, 5-10 minutes each time, to remove the initial Fmoc protecting group on the resin. Fmoc-Phe(tBu)-OH (Fmoc-protected phenylalanine, tBu-protected carboxyl) is mixed with DIC (diisopropyl carbodiimide) and Oxyma Pure (or HOBt) and added to the resin after activation. The reaction is usually carried out in DMF, stirring for 30-60 minutes. After the first amino acid reaction is complete, the next amino acid Pro is connected, and then the above steps b-f are repeated to connect DLeu, DMet, Pro, Gly, Arg, His, Cys, Leu, Arg, Pro, Arg, Cys, DArg, Dleu, DArg, DLys, respectively. Then the protecting group Fmoc on FmocPhe-OH is removed to expose the N-terminus. The last condensed amino acid is C 19 alkyl carboxylic acid, oligoethylene glycol and gamma glutamic acid, these amino acids do not have Fmoc group protection, so there is no need to remove Fmoc protection after condensation. The subsequent cleavage and purification steps are shown in Preparation Example 1.

[0816] MS characterization: mass spectrum shows the mass-to-charge ratio of the polypeptide, 3 target peaks: [M+5H] 5+ = 604.8, [M+4H] 4+ = 755.8, [M+3H] 3+ = 1007.5. The target peaks are detected at these 3 positions, so the purified polypeptide is indeed the title polypeptide sequence.

[0817] HPLC results show that the purity of the title polypeptide is 96.52%.

[0818] Preparation Example 7

[0819] Preparation of DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6)

[0820] The preparation process is as described in Preparation Example 1, and the required amino acid raw materials are cysteine, alanine, proline, arginine, leucine, cysteine, histidine, lysine, glycine, D-methionine, D-leucine, D-arginine, and phenylalanine. Other methods are as described in Preparation Example 1, and the resin with an amide linker is loaded into the SPPS reaction column after cleavage to automatically form a C-terminal amide. The resin is fully swollen with DMF (dimethylformamide) for at least 30 minutes. The resin is treated with a 20% piperidine / DMF solution twice for 5-10 minutes each time to remove the initial Fmoc protecting group on the resin. Fmoc-Phe(tBu)-OH (Fmoc-protected phenylalanine, tBu-protected carboxyl) is mixed with DIC (diisopropyl carbodiimide) and Oxyma Pure (or HOBt) and added to the resin after activation. The reaction is usually carried out in DMF, and stirring is performed for 30-60 minutes. After the first amino acid reaction is complete, the next amino acid Pro is connected, and then the above steps b-f are repeated to connect DLeu, DMet, Pro, Gly, Arg, His, Cys, Leu, Arg, Pro, Arg, Cys, DArg, Dleu, DArg, DLys, respectively. Then the protecting group Fmoc on FmocPhe-OH is removed to expose the N-terminus, and then the cleavage and purification steps are as shown in Preparation Example 1.

[0821] MS characterization: Mass spectrometry shows the mass-to-charge ratio of the polypeptide, 3 target peaks: [M+5H] 5+ = 422.1, [M+4H] 4+ = 527.4, [M+3H] 3+ = 702.9. The target peaks are detected at these three positions, so the purified product is indeed the title polypeptide sequence.

[0822] HPLC results show that the purity of the title polypeptide is 95.70%.

[0823] Preparation Example 8

[0824] C 19 alkyl

[0825] Preparation of -CO-γGlu-2xOEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6)

[0826] The preparation process is as described in Preparation Example 1, and the required amino acid raw materials are cysteine, alanine, proline, arginine, leucine, cysteine, histidine, lysine, glycine, D-methionine, D-leucine, D-arginine, phenylalanine, nonadecyl carboxylic acid / halide, gamma glutamic acid, oligoethylene glycol. Other methods are as described in Preparation Example 1, and the resin with an amide linker is taken, and after cleavage, the C-terminal amide is automatically formed. The resin is loaded into the SPPS reaction column. The resin is fully swollen with DMF (dimethylformamide) for at least 30 minutes. The resin is treated with a 20% piperidine / DMF solution twice for 5-10 minutes each time to remove the initial Fmoc protecting group on the resin. Fmoc-Phe(tBu)-OH (Fmoc-protected phenylalanine, tBu-protected carboxyl) is mixed with DIC (diisopropyl carbodiimide) and Oxyma Pure (or HOBt) and activated and added to the resin. The reaction is usually carried out in DMF, and stirring is carried out for 30-60 minutes. After the first amino acid reaction is complete, the next amino acid Pro is connected, and then the above steps b-f are repeated to connect DLeu, DMet, Pro, Gly, Arg, His, Cys, Leu, Arg, Pro, Arg, Cys, DArg, Dleu, DArg, DLys, respectively. Then the protecting group Fmoc on FmocPhe-OH is removed to expose the N-terminus. The last condensed amino acid is C19 alkyl carboxylic acid, oligoethylene glycol, and gamma glutamic acid, and these amino acids do not have Fmoc group protection, so after condensation, there is no need to remove the Fmoc protection. The subsequent cleavage and purification steps are as shown in Preparation Example 1.

[0827] MS characterization: Mass spectrum shows the mass-to-charge ratio of the polypeptide, 3 target peaks: [M+5H] 5+ = 570.9, [M+4H] 4+ = 713.3, [M+3H] 3+ = 950.6. The target peaks are detected at these three positions, so the purified polypeptide is indeed the title polypeptide sequence.

[0828] HPLC results show that the purity of the title polypeptide is 96.20%.

[0829] Preparation Example 9

[0830] Preparation of Ac-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 4-9)

[0831] The preparation procedure was the same as that of Preparation Example 4.

[0832] MS characterization: Mass spectrum shows the mass-to-charge ratio of polypeptide, 3 target peaks: [M+5H] 5+ = 427.0, [M+4H] 4+ = 570.1, [M+3H] 3+ = 855.0. The target peak was detected at these 3 positions, thus, the purified product is indeed the polypeptide sequence of the title.

[0833] HPLC result shows that the purity of the polypeptide of the title is 95.10%.

[0834] Preparation Example 10

[0835] Preparation of Ac-Lys-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 4-9)

[0836] The preparation procedure was the same as that of Preparation Example 4.

[0837] MS characterization: Mass spectrum shows the mass-to-charge ratio of polypeptide, 3 target peaks: [M+5H] 5+ = 424.4, [M+4H] 4+ = 565.5, [M+3H] 3+ = 848.0. The target peak was detected at these 3 positions, thus, the purified product is indeed the polypeptide sequence of the title.

[0838] HPLC result shows that the purity of the polypeptide of the title is 95.22%.

[0839] Preparation Example 11

[0840] Preparation of Ac-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 3-8)

[0841] The preparation procedure was the same as that of Preparation Example 4.

[0842] MS characterization: Mass spectrum shows the mass-to-charge ratio of polypeptide, 3 target peaks: [M+5H] 5+= 424.4, [M+4H] 4+ = 565.5, [M+3H] 3+ = 847.4. The peaks of interest were detected at all three positions, thus, the purified was indeed the title polypeptide sequence.

[0843] HPLC results showed that the purity of the title polypeptide was 97.20%.

[0844] Preparation Example 12

[0845] Preparation of Ac-Lys-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 3-8)

[0846] The preparation procedure was the same as that of Preparation Example 4.

[0847] MS characterization: Mass spectrum showed the mass-to-charge ratio of the polypeptide, three peaks of interest: [M+5H] 5+ = 374.2, [M+4H] 4+ = 467.4, [M+4H] 3+ = 622.8. The peaks of interest were detected at all three positions, thus, the purified was indeed the title polypeptide sequence.

[0848] HPLC results showed that the purity of the title polypeptide was 98.67%.

[0849] Preparation Example 13

[0850] Preparation of Ac-Arg-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 3-8)

[0851] The preparation procedure was the same as that of Preparation Example 4.

[0852] MS characterization: Mass spectrum showed the mass-to-charge ratio of the polypeptide, three peaks of interest: [M+5H] 5+ = 428.1, [M+4H] 4+ = 534.7. The peaks of interest were detected at all two positions, thus, the purified was indeed the title polypeptide sequence.

[0853] HPLC results showed that the purity of the title polypeptide was 6.26%.

[0854] Preparation Example 14

[0855] Preparation of Ac-Lys-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 5-10)

[0856] The preparation procedure was the same as that of Preparation Example 4.

[0857] MS characterization: Mass spectrum shows the mass-to-charge ratio of polypeptide, 3 target peaks: [M+5H] 5+ = 428.1, [M+4H] 4+ = 534.7. The target peaks were detected in both positions, so the purified product was indeed the sequence of the title polypeptide.

[0858] HPLC results showed that the purity of the title polypeptide was 96.61%.

[0859] Preparation Example 15

[0860] Preparation of Ac-Leu-Arg-Gly-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 5-10)

[0861] The preparation procedure was the same as that of Preparation Example 4.

[0862] MS characterization: Mass spectrum shows the mass-to-charge ratio of polypeptide, 3 target peaks: [M+5H] 5+ = 413.8, [M+4H] 4+ = 516.9. The target peaks were detected in both positions, so the purified product was indeed the sequence of the title polypeptide.

[0863] HPLC results showed that the purity of the title polypeptide was 96.18%.

[0864] Preparation Example 16

[0865] Preparation of Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 4-9)

[0866] The preparation procedure was the same as that of Preparation Example 4.

[0867] MS characterization: Mass spectrum shows the mass-to-charge ratio of polypeptide, 3 target peaks: [M+5H] 5+ = 422.1, [M+4H] 4+= 527.2, [M+3H] 3+ = 702.7. The peaks of interest were detected in all 3 positions, thus, the purified was indeed the title polypeptide sequence.

[0868] HPLC results showed that the purity of the title polypeptide was 96.27%.

[0869] Preparation Example 17

[0870] Preparation of Ac-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 4-9)

[0871] The preparation procedure was the same as that in Preparation Example 4.

[0872] MS characterization: mass spectrum showed the mass-to-charge ratio of the polypeptide, 4 peaks of interest: [M+5H] 5+ = 430.4, [M+4H] 4+ = 537.8, [M+3H] 3+ = 716.6. The peaks of interest were detected in all 4 positions, thus, the purified was indeed the title polypeptide sequence.

[0873] HPLC results showed that the purity of the title polypeptide was 96.75%.

[0874] Preparation Example 18

[0875] Preparation of Ac-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2 (Disulfide bridge: 4-9) (Disulfide bridge: 4-9)

[0876] The preparation procedure was the same as that in Preparation Example 4.

[0877] MS characterization: mass spectrum showed the mass-to-charge ratio of the polypeptide, 3 peaks of interest: [M+5H] 5+ = 424.2, [M+4H] 4+ = 566.0, [M+3H] 3+ = 847.4. The peaks of interest were detected in all 3 positions, thus, the purified was indeed the title polypeptide sequence.

[0878] HPLC results showed that the purity of the title polypeptide was 95.40%.

[0879] Preparation Example 19

[0880] COOHC 16 alkyl

[0881] Preparation of -CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge: 4-9)

[0882] The preparation procedure is the same as that of Preparation Example 4.

[0883] MS characterization: Mass spectrum shows the mass-to-charge ratio of the polypeptide, 3 target peaks: [M+5H] 5+ = 496.0, [M+4H] 4+ = 619.7, [M+3H] 3+ = 826. In these 4 positions, the target peaks are detected, thus, the purified product is indeed the polypeptide sequence of the title.

[0884] HPLC result shows that the purity of the polypeptide of the title is 95.28%.

[0885] Preparation Example 20

[0886] COOHC 18 alkyl

[0887] Preparation of -CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-GPF-NH2(Disulfide bridge: 4-9)

[0888] The preparation procedure is the same as that of Preparation Example 4.

[0889] MS characterization: Mass spectrum shows the mass-to-charge ratio of the polypeptide, 4 target peaks: [M+5H] 6+ = 418.1, [M+5H] 5+ = 501.5, [M+4H] 4+ = 626.8, [M+3H] 3+ = 835.2, [M+4H] 2+ = 1252.3. In these 5 positions, the target peaks are detected, thus, the purified product is indeed the polypeptide sequence of the title.

[0890] HPLC result shows that the purity of the polypeptide of the title is 96.48%.

[0891] Preparation Example 21

[0892] COOHC 16 alkyl

[0893] Preparation of -CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-GPF-NH2 (Disulfide bridge: 4-9)

[0894] The preparation procedure is the same as that of Preparation Example 4.

[0895] MS characterization: Mass spectrum shows the mass to charge ratio of the polypeptide, 4 target peaks: [M+5H] 5+ = 554.0, [M+4H] 4+ = 692.3, [M+3H] 3+ = 922.7, [M+5H] 6+ = 1383.8. The target peaks were detected in all the 4 positions, thus, the purified product is indeed the polypeptide sequence of the title.

[0896] HPLC result shows that the purity of the polypeptide of the title is 96.51%.

[0897] Preparation Example 22

[0898] COOHC 18 alkyl

[0899] Preparation of -CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-GPF-NH2 (Disulfide bridge: 4-9)

[0900] The preparation procedure is the same as that of Preparation Example 4.

[0901] MS characterization: Mass spectrum shows the mass to charge ratio of the polypeptide, 4 target peaks: [M+5H] 6+ = 559.7, [M+5H] 5+ = 699.33, [M+4H] 4+ = 932.1, [M+3H] 3+ = 1397.6. The target peaks were detected in all the 4 positions, thus, the purified product is indeed the polypeptide sequence of the title.

[0902] HPLC result shows that the purity of the polypeptide of the title is 95.84%.

[0903] Preparation Example 23

[0904] COOHC 16 alkyl

[0905] Preparation of -CO-γGlu-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-GPF-NH2 (Disulfide bridge: 4-9)

[0906] The preparation procedure was the same as that of Preparation Example 4.

[0907] MS characterization: Mass spectrum shows the mass-to-charge ratio of the polypeptide, 4 target peaks: [M+5H] 5+ = 521.8, [M+5H] 4+ = 652.0, [M+4H] 3+ = 868.8, The target peaks were detected at these 4 positions, thus, the purified product is indeed the polypeptide sequence of the title.

[0908] HPLC results showed that the purity of the polypeptide of the title was 96.37%.

[0909] Preparation Example 24

[0910] COOHC 18 alkyl

[0911] Preparation of -CO-γGlu-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-GPF-NH2 (Disulfide bridge: 4-9)

[0912] The preparation procedure was the same as that of Preparation Example 4.

[0913] MS characterization: Mass spectrum shows the mass-to-charge ratio of the polypeptide, 4 target peaks: [M+5H] 5+ = 527.4, [M+4H] 4+ = 659.0, [M+3H] 3+ = 878.3, [M+3H] 2+ = 1316.8. The target peaks were detected at these 4 positions, thus, the purified product is indeed the polypeptide sequence of the title.

[0914] HPLC results showed that the purity of the polypeptide of the title was 95.76%.

[0915] Preparation Example 25

[0916] COOHC 18 alkyl

[0917] Preparation of -CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2 (Disulfide bridge: 4-9)

[0918] Preparation procedure was same as that of Preparation Example 4.

[0919] MS characterization: Mass spectrum shows the mass to charge ratio of the polypeptide, 4 target peaks: [M+5H] 5+ = 495.9, [M+5H] 4+ = 619.6, [M+4H] 3+ = 826.0, [M+3H] 2+ = 1283.3. The target peaks were detected in all the 4 positions, thus, the purified product was indeed the polypeptide sequence of the title.

[0920] HPLC result showed that the purity of the polypeptide of the title was 95.49%.

[0921] Preparation Example 26

[0922] COOHC 16 alkyl

[0923] Preparation of -CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2 (Disulfide bridge: 4-9)

[0924] Preparation procedure was same as that of Preparation Example 4.

[0925] MS characterization: Mass spectrum shows the mass to charge ratio of the polypeptide, 4 target peaks: [M+5H] 5+ = 551.6, [M+4H] 4+ = 689.2, [M+3H] 3+ = 918.6, [M+3H] 2+ = 1377.3. The target peaks were detected in all the 4 positions, thus, the purified product was indeed the polypeptide sequence of the title.

[0926] HPLC result showed that the purity of the polypeptide of the title was 95.85%.

[0927] Preparation Example 26

[0928] COOHC 18 alkyl

[0929] Preparation of -CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His- Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2 (Disulfide bridge: 4-9)

[0930] Preparation procedure was same as that of Preparation Example 4.

[0931] MS characterization: Mass spectrum shows the mass to charge ratio of the polypeptide, 3 target peaks: [M+5H] 5+ = 554.1, [M+4H] 4+ = 692.3, [M+3H] 3+ = 922.7. The target peak was detected at all the 3 positions, thus, the purified product is indeed the polypeptide sequence of the title.

[0932] HPLC result showed that the purity of the polypeptide of the title was 97.32%.

[0933] Preparation Example 27

[0934] COOHC 18 alkyl

[0935] Preparation of -CO-γGlu-γGlu-DArg-DLeu-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His- Arg-Gly-Pro-DMet-DLeu-PF-NH2 (Disulfide bridge: 5-10)

[0936] Preparation procedure was same as that of Preparation Example 4.

[0937] MS characterization: Mass spectrum shows the mass to charge ratio of the polypeptide, 4 target peaks: [M+5H] 5+ = 552.8, [M+4H] 4+ = 690.9, [M+3H] 3+ = 920.7, [M+3H] 2+ = 1380.4. The target peak was detected at all the 4 positions, thus, the purified product is indeed the polypeptide sequence of the title.

[0938] HPLC result showed that the purity of the polypeptide of the title was 95.23%.

[0939] Preparation Example 28

[0940] COOHC 18 alkyl

[0941] Preparation of -CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-Cys-Arg-Pro-Arg- Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-PF-NH2 (Disulfide bridge: 5-10)

[0942] The preparation procedure was the same as that of Preparation Example 4.

[0943] MS characterization: Mass spectrum shows the mass-to-charge ratio of the polypeptide, 3 target peaks: [M+5H] 6+ = 487.7, [M+5H] 5+ = 585.1, [M+4H] 4+ = 731.0, [M+3H] 3+ = 974.4. The target peaks were detected at these 3 positions, so the purified product was indeed the polypeptide sequence of the title.

[0944] HPLC results showed that the purity of the title polypeptide was 95.72%.

[0945] Preparation Example 29

[0946] COOHC 18 alkyl

[0947] Preparation of -CO-γGlu-γGlu-DArg-DLeu-DArg-DAla-DArg-Cys-Arg-Pro-Arg- Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-PF-NH2 (Disulfide bridge: 6-11)

[0948] The preparation procedure was the same as that of Preparation Example 4.

[0949] MS characterization: Mass spectrum shows the mass-to-charge ratio of the polypeptide, 3 target peaks: [M+5H] 5+ = 604.2, [M+4H] 4+ = 760.3, [M+3H] 3+ = 1012.2. The target peaks were detected at these 3 positions, so the purified product was indeed the polypeptide sequence of the title.

[0950] HPLC results showed that the purity of the title polypeptide was 97.32%.

[0951] Preparation Example 30

[0952] COOHC 18 alkyl

[0953] Preparation of -CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-DArg-Cys-Arg-Pro-Arg- Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-PF-NH2(Disulfide bridge: 6-11)

[0954] The preparation procedure was the same as that of Preparation Example 4.

[0955] MS characterization: Mass spectrum shows the mass-to-charge ratio of the polypeptide, 3 target peaks: [M+5H] 5+ = 604.5, [M+4H] 4+ = 756.2, [M+3H] 3+ = 1002.0. The target peaks were detected at these 3 positions, thus, the purified product was indeed the polypeptide sequence of the title.

[0956] HPLC results showed that the purity of the polypeptide of the title was 97.32%.

[0957] Preparation Example 31

[0958] COOHC 18 alkyl

[0959] Preparation of -CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-DArg-DAla-Cys-Arg-Pro-Arg- Leu-Arg-Arg-Gly-Pro-DMet-DLeu-PF-NH2(Disulfide bridge: 7-12)

[0960] The preparation procedure was the same as that of Preparation Example 4.

[0961] MS characterization: Mass spectrum shows the mass-to-charge ratio of the polypeptide, 5 target peaks: [M+5H] 6+ = 525.6, [M+5H] 5+ = 630.6, [M+4H] 4+ = 788.1, [M+3H] 3+ = 1050.2, [M+5H] 2+ = 1575.1, The target peaks were detected at these 3 positions, thus, the purified product was indeed the polypeptide sequence of the title.

[0962] HPLC results showed that the purity of the polypeptide of the title was 95.32%.

[0963] Biological Example

[0964] 1. Nanobit experiment:

[0965] Experimental principle: Binary Technology (NanoBiT) is a structure-complementary reporter gene technology for detecting protein interaction research. The system consists of 2 subunits, Large BiT (LgBiT; 18 kDa) and Small BiT (SmBiT; 11 amino acids), which can be expressed in fusion with target proteins. The LgBiT and SmBiT subunits have been optimized to have good stability and minimized self-association. When the fusion target protein with the 2 subunits interacts, the 2 subunits will bind to each other to form an active enzyme, and a bright luminescent signal will be generated. We linked the Gα protein to LgBiT and the Gγ to SmBiT, and cloned the receptor gene into the pcDNA3.1 vector. The ability of the ligand to activate the receptor-coupled G protein was reflected by detecting the dissociation of Gα and Gγ.

[0966] Experimental method: The above plasmids were co-transfected into 6-well plates of cultured HEK293T cells for expression. After 24 hours of cell transfection, the culture medium was replaced with complete culture medium containing 10% FBS overnight. After 24-48 hours of cell transfection, the transfected 6-well plate was taken out, washed once with PBS, and the cells were resuspended with 3 ml of DMEM complete medium after 1 min of trypsin digestion. The resuspended cells were seeded into a 96-well plate and cultured overnight. The next day, the 96-well plate was taken out and washed once with D-PBS. First, incubate with 400a substrate prepared with HBSS for 50 min, read the baseline signal, then add different concentrations of ligand and mix, then read the signal after ligand stimulation. The ligand-induced signal was normalized to the baseline signal to show the dissociation reaction of G protein.

[0967] 2. BRET experiment:

[0968] Experimental principle: BRET experiment is to study the interaction between proteins by labeling the fusion protein of bioluminescent donor such as Renilla luciferase (Rluc) or fluorescent receptor such as enhanced green or yellow fluorescent protein (EGFP or EYFP). When the interacting parts are co-expressed in cells, in the presence of substrate coelenterazine, the protein distance is close enough (within 100 A), and energy transfer from Rluc to EYFP will occur. BRET is an ideal tool for studying receptor protein interactions involving all GPCR functions and regulation by agonists and antagonists due to the strict distance between donor and acceptor molecules.

[0969] Experimental method:

[0970] ​BRET method was used to detect the recruitment of β-arrestin. APLNR receptor C terminal fusion Rluc8 fragment, and the Venus fragment was inserted into the N terminal of β-arrestin2, respectively cloned into pBiT1.1 plasmid. The above-mentioned plasmid was co-transfected into 6-well plates of cultured HEK293T cells for expression, and the culture medium was replaced with complete culture medium containing 10% FBS overnight after 24 hours of cell transfection. After 24-48 hours of cell transfection, the transfected 6-well plate was taken out, washed once with PBS, and the cells were resuspended with 1ml trypsin for 1min and then resuspended with 3ml DMEM complete culture medium. The resuspended cells were inoculated into a 96-well plate and cultured overnight. The next day, the 96-well plate was taken out, washed once with D-PBS, and 40μl HBSS was added, then different concentrations of ligand (5μl) were used to stimulate transfected cells for 40min. Subsequently, the luciferase substrate coelenterazine 400h was added, and the BRET signal was detected: Venus (520-560nm), RLuc8 (460-485nm).

[0971] 3. Protein purification

[0972] Freeze-EM samples of APLNR-Gil bound apelin-, MM07-complexes were prepared. We cloned human wild type (WT) APLNR, Gil-Gp, scfv16 genes into pFastBacl vector (Invitrogen) and expressed in Sf9 insect cells at 1 : 1 : 1. To purify APLNR-Gil complexes, cell pellets were suspended in buffer containing 20 mM HEPES pH 7.5, 2 mM MgCl2, 100 mM NaCl and protease inhibitor cocktail (Bimake), followed by addition of 1.2 mg scFv16 antibody, 12 mU / ml apyrase (Sigma) and agonist (50 mM apelin, 50 mM MM07). The mixture was incubated at room temperature for 1 hour to form complexes. Then 0.5% (w / v) dodecyl maltose neopentyl glycol and 0.1% (w / v) hemi-succinyl cholesteryl were added, 4 °C incubation for 2 h. After incubation with MBP (NEB), the protein was eluted with 20 mM maltose. TEV protease was then added for 1 hour to remove MBP protein. The eluate was concentrated with 100 kDa concentrator tubes, the sample was injected into a Superose 6 increase 10 / 300 GL column (GE Healthcare) with running buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 0.00075% dodecyl maltose neopentyl glycol, 0.0002% (w / v) hemi-acetyl cholesteryl and 0.00025% (w / v) glycoside, agonist (50 mM apelin, 50 mM MM07). The peak fractions of interest were collected, concentrated to 10-15 mg / ml, and subjected to electron microscopy experiments.

[0973] 4. Data collection and structure resolution

[0974] For APLNR-Gil complexes, cryo-EM data collection was performed on a Titan Krios at 300 kV acceleration voltage. Micrographs were recorded in super-resolution mode using a Gatan K2 detector using SerialEM software (Schorb, Haberbosch et al. 2019) with pixel size of Image stacks were obtained at a dose rate of ~8.0 electrons / A2 / second with underfocus values ranging from -1.0 to -2.5 microns. Total exposure time was 8 seconds with 40 frames recorded per micrograph. A total of 2520 apelin-bound complex micrographs were collected. For MM07 complexes, cryo-EM data collection was performed on a Titan Krios at 300 kV acceleration voltage. Micrographs were recorded in super-resolution mode using a Gatan K2 detector using SerialEM software (Schorb, Haberbosch et al. 2019) with pixel size of A total of 4454 micrographs were collected at a dose rate of 21.3 electrons / Å2 / sec with underfocus ranging from -0.8 to -1.8 microns. The total exposure time was 2.35 sec and 36 frames were recorded for each micrograph. The collected data were processed using RELION 3.1 and CryoSPARC v3.1. The model was docked to the cryo-EM density map using UCSF Chimera (Pettersen, Goddard et al. 2004). The final model was refined using the “cryo-EM” module in Phenix. The structural figures were drawn using UCSF Chimera and UCSF Chimera X software.

[0975] Example 1

[0976] Structure determination of APLNR-G protein complex

[0977] The amino acid sequence of human APLNR is shown in SEQ ID No. 1 (NCBI sequence number: NP_005152.1), and the coding gene sequence is shown in SEQ ID No. 2 (NCBI sequence number: NM_005161.6). The G protein is human Gi1 (Gi1 is a subtype of Gi protein), and the amino acid sequence is shown in SEQ ID No. 3 (NCBI sequence number: NP_001027619.1), and the coding gene sequence is shown in SEQ ID No. 4 (NCBI sequence number: NM_001032447.1).

[0978] To investigate the ligand recognition mechanism of APLNR, we determined the cryo-EM structures of APLNR-Gi complex activated by the equilibrium agonist apelin and the G protein-biased agonist MM07 (CAS number: 1876450-21-3) at resolutions of 3.0 Å and 3.1 Å, respectively. and

[0979] First, stable apelin-13-APLNR-Gi1 (apelin-13 complex with APLNR and Gi1) and MM07-APLNR-Gi1 (MM07 complex with APLNR and Gi1) protein complexes were purified by expressing in insect expression system with baculovirus and stabilizing G protein trimer with the help of scFv16 antibody (laboratory expression and purification, amino acid sequence shown in SEQ ID No. 5). Then, data collection was performed using 300KV cryo-EM. Data processing and three-dimensional reconstruction were performed using RELION software, and modeling and structure refinement were performed using WINCOOT and PHENIX software to obtain high-resolution ternary complex structures.

[0980] The experimental results are shown in Figure 1. The results indicate that we have resolved the high-resolution cryo-electron microscopy structure of the apelin-APLNR-Gi1 complex (Figure 1, A and B), allowing for a good analysis of the interaction between the ligand and receptor. We overexpressed the APLNR receptor in HEK293 cells and used nanobit assays to detect the ability of apelin and MM07 to activate APLNR and recruit G proteins, and a Bret assay to detect the ability of the ligand to activate arrestin. The results are shown in Figure 1, C, where the EC50 of apelin activating G protein signaling is 9.793 × 10⁻⁶. -10 M, recruiting β-arrestin EC 50 It is 8.688×10 -9 MM07 activates the EC of G protein signaling. 50 1.377×10 -8 M, recruiting β-arrestin EC 50 It is 5.495×10 -6 Compared to apelin, MM07's ability to activate arrestin was nearly 1000-fold weaker, while its G protein activity was about 10-fold weaker than apelin's. These results indicate that MM07 is a G protein-biased agonist, but still retains considerable arrestin activity.

[0981] Example 2

[0982] Analysis of APLNR biased signal transduction mechanism

[0983] By comparing the differences in the APLNR binding pocket activated by two ligands, the specific amino acid sites for binding to APLNR by different agonists were identified, elucidating the recognition and activation mechanisms of the endogenous peptide apelin and the G protein-biased agonist MM07. Mutations were performed on key amino acid sites in APLNR involved in ligand recognition, and nanobit and BRET assays were used to detect the ligand-activated downstream G protein and β-arrestin activities, respectively. This determined the effect of point mutations on the ability of APLNR to bind to downstream G proteins and β-arrestin under different ligand effects, thus elucidating the biased signal transduction mechanism.

[0984] The results are shown in Figure 2. The residues of the ligand binding pocket of the APLNR structure bound by apelin (A) and MM07 (B) are shown with surface representation of the density of Ml 1 and F13 of the polypeptide ligand and the residues on the receptor that interact with these two amino acids. The functional experiment results show that mutation of the key amino acids located in the "twin hotspots" affects the G protein or β-arrestin activity (the β-arrestin activity is reduced more than the G protein after mutation of the amino acids shown in C, E of Figure 2, the receptor is biased to bind the G protein after mutation; the G protein activity is reduced more than the β-arrestin after mutation of the amino acids shown in D, F of Figure 2, the receptor is biased to bind the β-arrestin), indicating that the two pockets "twin hotspots" on the receptor are the key to affecting the selectivity of APLNR to downstream signaling molecules. Ml 1 and F13 of apelin and MM07 respectively insert the two pockets and interact with the key amino acid residues of the "twin hotspots". Therefore, the Met at position 11 and the Phe at position 13 that change the interaction with the receptor help to design the G protein biased polypeptide agonist.

[0985] Example 3

[0986] The detection method is as described in Example 1, we analyzed the agonist activated receptor binding G protein complex structure, analyzed the ligand binding pocket, we determined all the key amino acid residues that affect the binding of the ligand to the receptor as shown in Figure 27, including: Y35 1.39 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , I109 3.32 , F110 3.33 , M113 3.36 , R168 4.64 , M183 ECL2 , Y185 ECL2 , E198 5.35 , Y264 6.51 , K268 6.55 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , T295 7.39 , Y299 7.43 , M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y351.39 T295 7.39 Y299 7.49 F78 2.53 W85 2.60 Y88 2.63 Q180 ECL2 and P292 7.36 .

[0987] We used the same signal detection method as in Example 2 to detect Gα and Gβγ dissociation by nanobit to determine the effect of all amino acid site mutations interacting with apelin and MM07 on receptor activity, and found that M183 ECL2 F110 3.33 I109 3.32 K265 6.55 Y35 1.39 Y299 7.49 W85 2.60 Y88 2.63 Y93 ECL1 R168 4.64 Y185 ECL2 E198 5.35 Y271 6.58 M288 7.32 P163 4.59 L201 5.38 V164 4.60 S205 5.42 P292 7.36 M288 7.32 Q180 ECL2 Y182 ECL2 are key amino acid residues affecting receptor interaction, as shown in the following table ΔpEC 50 and Emax, these amino acid sites significantly weaken APLNR-mediated G protein signaling after alanine mutation, EC 50 relative to wild type (WT), so these amino acids are key amino acids for receptor and ligand interaction.

[0988] Data are expressed as mean ± SEM. *P <0.05, **P <0.01, ***P <0.001 by one-way ANOVA, ns: not significant, and Dunnett's multiple comparison test compared with the control group. ND, complete loss of activity, EC 50 .

[0989] Table 2. Gα-Gγ dissociation induced by ligands in wild type (WT) and APLNR mutants

[0990] We also identified the key amino acid residues that affect the receptor bias, as shown in Figure 2 and Table 3, M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 are the key amino acids that affect the bias, and the difference in the interaction of these amino acids directly affects the bias of the receptor to G protein (Table 2) and β-arrestin (Table 3), which is the basis for our polypeptide design.

[0991] Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA, ns: not significant, followed by Dunnett's multiple comparison test compared with the control group.

[0992] Table 3. Ligand-induced β-arrestin recruitment of wild-type (WT) and APLNR mutants

[0993] Example 4

[0994] In order to obtain a stable polypeptide sequence with activity, we used various strategies for optimization, such as adding fatty acid modification at the N-terminus, PEG, fusion protein, etc. The polypeptide mutants of the present disclosure were prepared using the methods described in Preparation Examples 1 to 3, and the functional experiment results are shown in Table 4. The β-arrestin activity is lost, the G protein activity is retained, and it is completely biased to G protein.

[0995] Table 4. G protein and β-arrestin activity and bias statistics of polypeptide sequences

[0996] Example 5

[0997] Pharmacokinetic property test in rat model

[0998] Subcutaneous administration: The rats were fasted overnight, and the test compound was dissolved in PBS (concentration 0.2 mg / mL) for administration at a dose of 1 mg / kg. At 0.5 h, 2 h, 8 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h after administration, blood (0.2 mL each time) was continuously taken from the jugular vein and placed in EP tubes containing EDTA-K2. After centrifugation at 4000 g for 5 min at 4°, the upper plasma was taken and stored at -80° for analysis by LC-MS / MS. According to the blood concentration-time data obtained by testing, the pharmacokinetic parameters were calculated using WinNonlin software.

[0999] Pharmacokinetic property test in cynomolgus monkey model

[1000] Subcutaneous administration: The cynomolgus monkeys were fasted overnight, and the test compound was dissolved in PBS (concentration 0.5 mg / mL) for administration at a dose of 0.5 mg / kg. At 0.5 h, 2 h, 8 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h, 240 h, 336 h, 504 h after administration, blood (0.3 mL each time) was continuously taken from the peripheral vein and placed in EP tubes containing EDTA-K2. After centrifugation at 2000 x g for 10 min at 2-8°, the upper plasma was taken and stored at -80° for analysis by LC-MS / MS. According to the blood concentration-time data obtained by testing, the pharmacokinetic parameters were calculated using WinNonlin software.

[1001] Biological Example 5

[1002] DIO mouse modeling and efficacy detection

[1003] Diet-induced obese (DIO) mice were generated by placing 5-week-old C57BL / 6 mice on a high-fat diet (60 kcal% fat) for about 19 weeks. The initial body weight was recorded before the high-fat diet feeding, and then the body weight was monitored once a week (typical DIO mice weighed between 40 and 50 grams). The body weight, body composition, and food intake were measured for randomization. After randomization, the mice were treated in the following groups (n=8): a total of 34 days of administration, daily administration.

[1004] Group 1: Tirzepatide (0.05 mpk, subcutaneous injection) + Sequence 216 (5 mpk, subcutaneous injection) combination in DIO mice

[1005] Group 2: Tirzepatide (0.05 mpk, subcutaneous injection) + BGE-105 (0.01 mpk, gavage) combination in DIO mice

[1006] Group 3: DIO mice with blank control (subcutaneous injection of equal volume of PBS solution)

[1007] Group 4: DIO mice with GLP-1 receptor modulator, tirzepatide (0.05 mpk, subcutaneous injection)

[1008] The main evaluation indexes include:

[1009] Daily cage-side observation;

[1010] Bi-weekly body weight and food intake statistics;

[1011] Weekly body composition measurement, including muscle mass, muscle content, fat mass, fat content, etc.; body composition is compared by (baseline, week 2, week 3 and week 4) or at the end point after the completion of administration using muscle weight / fat tissue weight ratio.

[1012] After the end of the experiment, the end point body composition is collected by histological dissection of muscle and fat content.

[1013] On the day of dissection, plasma and tissues / organs are collected. The tissues are weighed and further tested.

[1014] Tissues: half of the tissue samples are frozen for molecular biology analysis, and the other half is fixed in 10% neutral buffered formalin (or embedded in OTC) for histological analysis to measure and quantify fat: inguinal fat, perirenal fat, epididymal fat, brown fat; muscle: soleus, extensor digitorum longus, tibialis anterior, gastrocnemius; heart.

[1015] FIGS. 4A and 4B show the body weight change of each group of DIO mice during the administration period (28 days). All tirzepatide treatment groups show a significant decrease in body weight. Group 1: DIO mice treated with tirzepatide (0.05 mpk, subcutaneous injection) + sequence 216 (5 mpk, subcutaneous injection) combination shows a more obvious trend of body weight decrease. FIGS. 4C and 4D show the Q2D food intake and cumulative food intake change of each group of DIO mice during the administration period (28 days). All tirzepatide treatment groups show a significant decrease in food intake.

[1016] FIGS. 5A to 5C show the muscle content change of each group of DIO mice before administration, 1 week, 2 weeks, 3 weeks and 4 weeks after administration. Compared with tirzepatide administration alone, the combined dose of sequence 216 and tirzepatide significantly increased muscle content during the 21st to 28th day.

[1017] Figures 6A-6C show the change in fat content in each group of DIO mice at pre-dose, 1 week, 2 weeks, 3 weeks, and 4 weeks of dosing. The use of the combination of SEQ ID NO: 216 and tirzepatide dosing significantly reduced fat content during the period of days 21-28 compared to treatment with tirzepatide dosing alone.

[1018] Figures 7A-7C show the change in fat mass in each group of DIO mice at pre-dose, 1 week, 2 weeks, 3 weeks, and 4 weeks of dosing. The use of the combination of SEQ ID NO: 216 and tirzepatide dosing significantly reduced fat content during the period of days 21-28 compared to treatment with tirzepatide dosing alone.

[1019] Figures 8A-8B show the change in muscle mass in each group of DIO mice at pre-dose, 1 week, 2 weeks, 3 weeks, and 4 weeks of dosing. The use of the combination of SEQ ID NO: 216 and tirzepatide dosing resulted in a rebound in muscle mass in the treatment group at day 21 compared to treatment with tirzepatide dosing alone.

[1020] Figures 9A-9B show the change in muscle mass / fat mass in each group of DIO mice at pre-dose, 1 week, 2 weeks, 3 weeks, and 4 weeks of dosing. The use of the combination of SEQ ID NO: 216 and tirzepatide dosing resulted in an increase in muscle / fat ratio in the mice receiving the combination compared to mice receiving tirzepatide alone.

[1021] Figures 10A-10C show the total muscle content of each group of dissected tissues at the end of dosing. The use of the combination of SEQ ID NO: 216 and tirzepatide dosing resulted in a significant increase in muscle tissue mass / fat tissue mass ratio in the mice receiving the combination compared to mice receiving tirzepatide alone.

[1022] The above results demonstrate that SEQ ID NO: 216 significantly enhances the weight loss effects of tirzepatide and overcomes the muscle loss side effects that can be caused by tirzepatide.

[1023] Figures 11A-11B show that mice receiving SEQ ID NO: 216 alone lost 11% of their body weight compared to the control group, without affecting appetite.

[1024] Figures 12A-12D show that mice receiving SEQ ID NO: 216 alone had a significant reduction in epididymal fat and a significant increase in brown fat compared to the control group. This demonstrates that treatment with SEQ ID NO: 216 reshapes the fat tissue composition of the mice, and that this can be used to exert its potential weight control or metabolic improvement effects by reducing energy storing white fat and increasing energy expending brown fat.

[1025] Results: Mice treated with tirzepatide and SEQ ID NO: 216 combination dosing (Treatment Group 1) resulted in weight loss while maintaining muscle function and reducing muscle loss.

[1026] Biological Example 6

[1027] Cardiomyocyte immunofluorescence: To observe the effect of Apelin and its analogs sequence 221, sequence 250, sequence 253 on the morphology of cardiomyocytes, immunofluorescence experiments were performed using rat cardiomyocyte cell line H9C2. Cells were treated with 10 μM for 36 hours on the basis of giving 1 μM PE (Phorbol 12-myristate 13-acetate) treatment or no treatment. After the end of treatment, DAPI was used to stain the cell nucleus with blue fluorescence, and phalloidin was used to stain the actin cytoskeleton with red fluorescence, and the cell size was photographed under a fluorescence microscope.

[1028] TAC modeling and drug administration: A pressure overload-induced heart failure model was established by aortic constriction (Transverse Aortic Constriction, TAC) operation. After the mice were anesthetized by inhaling 3% isoflurane, a thoracotomy was performed, and a 7-0 suture was used to ligate at the aortic arch (specific location: between the brachiocephalic artery and the left common carotid artery) and a needle was used to assist in forming a local stenosis, and then the needle was withdrawn. One week after the operation, the mice were given intraperitoneal injections of 1 mpk sequence 221, and the control group was injected with an equal volume of PBS buffer. Three and five weeks after administration, the mice were subjected to echocardiography to assess changes in cardiac structure and function.

[1029] Echocardiography: Using the Vevo2100 ultrasound system, the mice were anesthetized (1% isoflurane) and the parasternal short-axis view was obtained. The left ventricular internal diameter at end-diastole (LVIDd) and at end-systole (LVIDs) were measured. Ejection fraction (EF%) = [(LVIDd 3 -LVIDs 3 ) / LVIDd 3 ] x 100; Fractional shortening (FS%) = [(LVIDd-LVIDs) / LVIDd] x 100. The data were averaged over 5 consecutive cardiac cycles.

[1030] QPCR: The extracted heart samples were subjected to RNA extraction and reverse transcription to cDNA, and the expression levels of ANP, Myh7, and Myh6 were detected by QPCR. The amplification conditions were as follows: 95°C pre-denaturation for 30 seconds→35 cycles (95°C denaturation for 15 seconds→60°C annealing / extension for 30 seconds). The data were processed using the ΔΔCt method, and the average mRNA expression of at least 3 independent experiments was used as the result.

[1031] Heart section immunohistochemistry: The myocardial tissue of TAC model mice was fixed with 4% paraformaldehyde, labeled with Sirius red for collagen fibers, and stained with Alexa Fluor 488 conjugated WGA for cell boundaries. Images were taken by Leica TCS-SP8 confocal microscope (20x objective), and the cell surface area was quantified by NIH Image J software.

[1032] Figure 14 shows that Apelin, SEQ ID NO: 221, SEQ ID NO: 250 and SEQ ID NO: 253 all have inhibitory effects on PE-induced cardiomyocyte hypertrophy. However, Apelin can cause normal cardiomyocyte hypertrophy, in contrast, SEQ ID NO: 221, SEQ ID NO: 250 and SEQ ID NO: 253 have no pro-hypertrophic effect on normal cardiomyocytes.

[1033] Figure 15 shows that, within the five-week intervention period, the TAC+SEQ ID NO: 221 group significantly improved the cardiac function indicators of heart failure mice: inhibited the increase in left ventricular internal diameter at end-systole (LVIDs) and left ventricular mass (LV Mass), and prevented the decrease in ejection fraction (EF%) and fractional shortening (FS%).

[1034] Figure 16 shows that SEQ ID NO: 221, SEQ ID NO: 250 and SEQ ID NO: 253 significantly inhibited cardiomyocyte hypertrophy.

[1035] Figure 17 shows that these drugs also significantly reversed TAC-induced overall cardiac hypertrophy, with SEQ ID NO: 221 showing the best effect when administered daily and every three days.

[1036] Figure 18 shows that, at the molecular level, the SEQ ID NO: 221 administration group effectively reduced the expression of atrial natriuretic peptide (ANP) and inhibited the increase in the Myh7 / Myh6 ratio caused by TAC.

[1037] The Sirius red staining results in Figure 19 show the degree of myocardial fibrosis. By calculating the ratio of fibrosis area to cell area, the level of myocardial fibrosis was quantified. The results show that the myocardial fibrosis area of the SEQ ID NO: 221 daily administration and every three days administration groups and the SEQ ID NO: 253 administration group is significantly smaller than that of the TAC group, indicating that SEQ ID NO: 221 and SEQ ID NO: 253 treatment significantly inhibited the occurrence of cardiac fibrosis.

[1038] In the present disclosure, relational terms such as first and second and the like can merely be used to distinguish one entity or action from another, without necessarily requiring or implying that there is any such actual relationship or order between or among the entities or actions.

[1039] From the foregoing it will be appreciated that, although specific embodiments of the disclosure have been described herein for the purpose of illustration, various modifications and changes in the described embodiments can be made by those skilled in the art without departing from the spirit and scope of the disclosure. Such modifications and changes are intended to fall within the scope of the appended claims.

Claims

1. An Apelin-13 polypeptide mutant comprising (1) a polypeptide represented by general formula (I): aa1-Arg-Pro-Arg-Leu-b1-His-cc1-Gly-Pro-DMet-Gly-Pro-Phe General Formula (I) wherein, b1 is a side chain negatively charged amino acid, aa1 is selected from C 11-25 hydrocarbyl-γGlu, carboxyl-C 10-24 hydrocarbyl-γGlu, C 11-25 hydrocarbyl-γGlu-mxOEG, carboxyl-C 10-24 hydrocarbyl-γGlu-mxOEG, C 11-25 hydrocarbyl-γGlu-nxPEG, carboxyl-C 10-24 hydrocarbyl-γGlu-nxPEG or PEG, cc1 is selected from C 11-25 hydrocarbyl-γGlu-Lys, carboxyl-C 10-24 hydrocarbyl-γGlu-Lys, C 11-25 hydrocarbyl-mxOEG-γGlu-Lys, carboxyl-C 10-24 hydrocarbyl-mxOEG-γGlu-Lys or PEG-Lys, and the amino acid residues in positions 1, 6 form an amide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (I) and having the same function.

2. The Apelin-13 polypeptide mutant of claim 1, wherein said aa1 is selected from C 11-25 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu-2xOEG, carboxy-C 10-24 alkanoyl-yGlu-3xOEG or PEG.

3. The Apelin-13 polypeptide mutant of claim 1 or 2, wherein the b1 is selected from the group consisting of Glu and Asp.

4. The Apelin-13 polypeptide mutant of any one of claims 1 to 3, wherein the cc1 is selected from C 11-25 alkanoyl-γGlu-Lys, carboxy-C 10-24 alkanoyl-γGlu-Lys, carboxy-C 10-24 alkanoyl-2xOEG-γGlu-Lys, carboxy-C 10-24 alkanoyl-3xOEG-γGlu-Lys or PEG-Lys.

5. The Apelin-13 polypeptide mutant of any one of claims 1 to 4, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

6. The Apelin-13 polypeptide mutant of any one of claims 1 to 5, consisting of a polypeptide represented by general formula (I).

7. The Apelin-13 polypeptide mutant of any one of claims 1 to 6, wherein the polypeptide represented by general formula (I) is: C 15 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (amide bond at positions 1,6).

8. An Apelin-13 polypeptide mutant comprising (1) a polypeptide represented by general formula (II): aa2-Cys-a2-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe General Formula (II) wherein a2 is selected from an amino acid with a hydrophobic side chain or a polar uncharged side chain, aa2 is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxy-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxy-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues in positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5, n is an integer from 1 to 8; or (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (II) and having the same function.

9. The Apelin-13 polypeptide mutant of claim 8, wherein the a2 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn and Gin.

10. The Apelin-13 polypeptide mutant of claim 8 or 9, wherein said aa2 is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu-2xOEG, carboxy-C 10-24 alkanoyl-γGlu-3xOEG or PEG.

11. The Apelin-13 polypeptide mutant of any one of claims 8 to 10, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

12. The Apelin-13 polypeptide mutant of any one of claims 8 to 11, consisting of a polypeptide represented by general formula (II).

13. The Apelin-13 polypeptide mutant of any one of claims 8 to 12, wherein the polypeptide represented by general formula (II) is selected from the group consisting of: C 19 Alkyl-CO-γGlu-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-2xOEG-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOH-C 18 Alkyl-CO-γGlu-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOH-C 22 Alkyl-CO-γGlu-2xOEG-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG2-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG3-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG5-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and PEG8-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

14. An Apelin-13 polypeptide mutant comprising (1) a polypeptide represented by general formula (III-1): aa3-Arg-Pro-Arg-Leu-b3-His-cc3-Gly-Pro-DMet-Gly-Pro-Phe aa3-Cys-Arg-a3-Arg-b3-Cys-His-c3-Gly-Pro-d3-e3-f3-g3 Polypeptide represented by General Formula (III-1) wherein, a3 is selected from an amino acid with a hydrophobic side chain or a polar uncharged side chain, b3 is an amino acid with a hydrophobic side chain, c3 is an amino acid with a positively charged side chain, d3 and e3 are each independently selected from an amino acid with a hydrophobic side chain or Gly, f3 is an amino acid with a hydrophobic side chain or Pro, g3 is an amino acid with a hydrophobic side chain, aa3 is selected from C 11-25 alkanoyl-gamma Glu, carboxyl-C 10-24 alkanoyl-gamma Glu, C 11-25 alkanoyl-gamma Glu-mxOEG, carboxyl-C 10-24 alkanoyl-gamma Glu-mxOEG, C 11-25 alkanoyl-gamma Glu-nxPEG, carboxyl-C 10-24 alkanoyl-gamma Glu-nxPEG or PEG, and the cysteine residues in positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or (2) Polypeptide represented by General Formula (III-2): aa3'-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-a3' General Formula (III-2) wherein a3' is an amino acid with a hydrophobic side chain, aa3' is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxy-C 10-24 alkanoyl diacid-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxy-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5, n is an integer from 1 to 8; or (3) Polypeptide having at least 70% homology with the polypeptide represented by General Formula (III-1) or General Formula (III-2) and having the same function.

15. The Apelin-13 polypeptide mutant of claim 14, wherein the a3 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, and Gin.

16. The Apelin-13 polypeptide mutant of claim 14 or 15, wherein the b3 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

17. The Apelin-13 polypeptide mutant of any one of claims 14 to 16, wherein the c3 is selected from the group consisting of Lys and Arg.

18. The Apelin-13 polypeptide mutant of any one of claims 14 to 17, wherein the d3 and e3 are each independently selected from the group consisting of Ala, Val, lie, Leu, DMet, Phe, Tyr, Trp, and Gly.

19. The Apelin-13 polypeptide mutant of any one of claims 14 to 18, wherein the f3 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, and Pro.

20. The Apelin-13 polypeptide mutant of any one of claims 14 to 19, wherein the g3 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

21. The Apelin-13 polypeptide mutant of any one of claims 14 to 20, wherein the a3' is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

22. The Apelin-13 polypeptide mutant of any one of claims 14 to 21, wherein each of aa3 and aa3' is independently selected from C 11-25 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu-2xOEG, carboxy-C 10-24 alkanoyl-yGlu-3xOEG or PEG.

23. The Apelin-13 polypeptide mutant of any one of claims 14 to 22, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , 180 ECL2 , or 182 ECL2 .

24. The Apelin-13 polypeptide mutant of any one of claims 14 to 23, which consists of the polypeptide represented by General Formula (III-1) or the polypeptide represented by General Formula (III-2).

25. The Apelin-13 polypeptide mutant of any one of claims 14 to 24, wherein the polypeptide represented by General Formula (III-1) is selected from the group consisting of: C 15 Alkyl-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Leu (Disulfide bridge: 1-6); C 15 Alkyl-CO-γGlu-Cys-Arg-Trp-Lys-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Tyr- Phe (Disulfide bridge: 1-6); C 15 Alkyl-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and C 15 Alkyl-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

26. The Apelin-13 polypeptide mutant of any one of claims 14 to 24, wherein the polypeptide represented by General Formula (III-2) is selected from the group consisting of: C 15 Alkyl-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro- Trp (Disulfide bridge: 1-6).

27. An Apelin-13 polypeptide mutant comprising (1) a polypeptide represented by General Formula (IV): aa3'-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-a3' aa4-Cys-Arg-Pro-a4-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe General Formula (IV) wherein a4 is an amino acid with a hydrophobic side chain, aa3 is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxy-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxy-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues in positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5, n is an integer from 1 to 8; or (2) a polypeptide having at least 70% homology with the polypeptide represented by General Formula (IV) and having the same function.

28. The Apelin-13 polypeptide mutant of claim 27, wherein the a4 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

29. The Apelin-13 polypeptide mutant of claim 27 or 28, wherein said aa4 is selected from C 11-25 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu-2xOEG, carboxy-C 10-24 alkanoyl-yGlu-3xOEG or PEG.

30. The Apelin-13 polypeptide mutant of any one of claims 27 to 29, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , 180 ECL2 , or 182 ECL2 .

31. The Apelin-13 polypeptide mutant of any one of claims 27 to 30, consisting of a polypeptide represented by General Formula (IV).

32. The Apelin-13 polypeptide mutant of any one of claims 27 to 31, wherein the polypeptide represented by General Formula (IV) is selected from the group consisting of: C 19 Alkyl-CO-γGlu-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 22 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG2-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG3-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG5-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and PEG8-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

33. An Apelin-13 polypeptide mutant comprising (1) a polypeptide represented by General Formula (V-1): aa5-Cys-Arg-a5-Arg-b5-Cys-His-c5-Gly-Pro-d5-e5-f5-g5 General Formula (V-1) wherein a5 is selected from an amino acid with a hydrophobic side chain or Pro, b5 is an amino acid with a hydrophobic side chain, c5 is an amino acid with a positively charged side chain, d5 and e5 are each independently selected from an amino acid with a hydrophobic side chain or Gly, f5 is an amino acid with a hydrophobic side chain or Pro, g5 is an amino acid with a hydrophobic side chain, aa1 is selected from C 11-25 alkanoyl-gamma Glu, carboxy-C 10-24 alkanoyl-gamma Glu, C 11-25 alkanoyl-gamma Glu-mxOEG, carboxy-C 10-24 alkanoyl-gamma Glu-mxOEG, C 11-25 alkanoyl-gamma Glu-nxPEG, carboxy-C 10-24 alkanoyl-gamma Glu-nxPEG or PEG, and the cysteine residues in positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or (2) a polypeptide represented by General Formula (V-2): aa5’-Cys-Arg-a5’-Arg-b5’-Cys-His-c5’-Gly-Pro-Nal-Pro-d5’ General Formula (V-2) wherein a5' is selected from an amino acid having a hydrophobic side chain or Pro, b5' is an amino acid having a hydrophobic side chain, c5' is an amino acid with a positively charged side chain, d5' is an amino acid having a hydrophobic side chain, aa5' is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxy-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxy-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or (3) a polypeptide having at least 70% homology with the polypeptide represented by General Formula (V-1) or General Formula (V-2) and having the same function.

34. The Apelin-13 polypeptide mutant of claim 33, wherein the a5 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, and Pro.

35. The Apelin-13 polypeptide mutant of claim 33 or 34, wherein the b5 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

36. The Apelin-13 polypeptide mutant of any one of claims 33 to 35, wherein the c5 is selected from the group consisting of Lys and Arg.

37. The Apelin-13 polypeptide mutant of any one of claims 33 to 36, wherein the d5 and e5 are each independently selected from the group consisting of Ala, Val, lie, Leu, DMet, Phe, Tyr, Trp, and Gly.

38. The Apelin-13 polypeptide mutant of any one of claims 33 to 37, wherein the f5 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, and Pro.

39. The Apelin-13 polypeptide mutant of any one of claims 33 to 38, wherein the g5 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

40. The Apelin-13 polypeptide mutant of any one of claims 33 to 39, wherein said aa5 is selected from C 11-25 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu-2xOEG, carboxy-C 10-24 alkanoyl-yGlu-3xOEG or PEG.

41. The Apelin-13 polypeptide mutant of any one of claims 33 to 40, wherein the a5' is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, and Pro.

42. The Apelin-13 polypeptide mutant of any one of claims 33 to 41, wherein the b5' is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

43. The Apelin-13 polypeptide mutant of any one of claims 33 to 42, wherein the c5' is selected from the group consisting of Lys and Arg.

44. The Apelin-13 polypeptide mutant of any one of claims 33 to 43, wherein the d5' is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

45. The Apelin-13 polypeptide mutant of any one of claims 33 to 33, wherein the aa5’ is selected from C 11-25 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu-2xOEG, carboxy-C 10-24 alkanoyl-yGlu-3xOEG or PEG.

46. The Apelin-13 polypeptide mutant of any one of claims 33 to 45, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

47. The Apelin-13 polypeptide mutant of any one of claims 33 to 46, consisting of a polypeptide represented by Formula (V-1) or a polypeptide represented by Formula (V-2).

48. The Apelin-13 polypeptide mutant of any one of claims 33 to 47, the polypeptide represented by Formula (V-1) is selected from the group consisting of: C 17 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 19 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 21 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 16 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 16 Alkyl-CO-γGlu-OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 11 Alkyl CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 13 Alkyl CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 15 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 19 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 21 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 16 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-4xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and C 17 Alkyl-CO-γGlu-5xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

49. The Apelin-13 polypeptide mutant of any one of claims 33 to 47, the polypeptide represented by Formula (V-2) is selected from the group consisting of: C 19 Alk CO-Glu-Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe (Disulfide bridge: 1-6); C 17 Alk CO-Glu-2 x OEG-Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe (Disulfide bridge: 1-6); and PEG8-Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe (Disulfide bridge: 1-6).

50. An Apelin-13 polypeptide mutant comprising (1) a polypeptide represented by Formula (VII): aa7-Cys-Arg-Pro-Arg-Leu-Cys-a7-Lys-Gly-Pro-DMet-Gly-Pro-Phe Formula (VII) wherein a7 is selected from an amino acid having a hydrophobic side chain or a polar uncharged side chain, aa7 is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxy-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxy-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues in positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5, n is an integer from 1 to 8; or (2) a polypeptide having at least 70% homology to the polypeptide represented by Formula (VII) and having the same function.

51. The Apelin-13 polypeptide mutant of claim 50, wherein said a7 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, and Gin.

52. The Apelin-13 polypeptide mutant of claim 50 or 51, wherein said aa7 is selected from C 11-25 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu-2xOEG, carboxy-C 10-24 alkanoyl-yGlu-3xOEG or PEG.

53. The Apelin-13 polypeptide mutant of any one of claims 50 to 52, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

54. The Apelin-13 polypeptide mutant of any one of claims 50 to 53, consisting of a polypeptide represented by general formula (VII).

55. The Apelin-13 polypeptide mutant of any one of claims 50 to 54, wherein said polypeptide represented by general formula (VII) is selected from the group consisting of: C 19 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 22 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG2-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG3-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG5-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and PEG8-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe.

56. An Apelin-13 polypeptide mutant comprising (1) a polypeptide represented by general formula (VIII-1): aa8-Cys-Arg-a8-Arg-b8-Cys-His-c8-Gly-Pro-DMet-Gly-d8-e8 general formula (VIII-1) wherein a8is selected from an amino acid with a hydrophobic side chain or Pro, b8is an amino acid with a hydrophobic side chain, c8is selected from an amino acid with a hydrophobic side chain or an amino acid with a positively charged side chain, d8is an amino acid with a hydrophobic side chain or Pro, e8is an amino acid with a hydrophobic side chain, aa8is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxy-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxy-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues in positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or (2) a polypeptide represented by general formula (VIII-2): aa8’-Cys-Arg-Pro-Arg-Trp-Cys-His-a8’-Gly-Pro-Nal-Pro-Phe general formula (VIII-2) wherein a8' is a side chain positively charged amino acid, aa8' is selected from C 11-25 alkanoyl-γGlu, carboxyl-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxyl-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxyl-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5, n is an integer from 1 to 8; or (3) a polypeptide having at least 70% homology to the polypeptide represented by general formula (VIII-1) or general formula (VIII-2) and having the same function.

57. The Apelin-13 polypeptide mutant of claim 56, wherein said a8 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, and Pro.

58. The Apelin-13 polypeptide mutant of claim 56 or 57, wherein said b8 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

59. The Apelin-13 polypeptide mutant of any one of claims 56 to 58, wherein said c8 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Arg, and Lys.

60. The Apelin-13 polypeptide mutant of any one of claims 56 to 59, wherein the d8 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp and Pro.

61. The Apelin-13 polypeptide mutant of any one of claims 56 to 60, wherein the e8 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr and Trp.

62. The Apelin-13 polypeptide mutant of any one of claims 56-61, wherein said aa8 is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu-2xOEG, carboxy-C 10-24 alkanoyl-γGlu-3xOEG or PEG.

63. The Apelin-13 polypeptide mutant of any one of claims 56 to 62, wherein the a8' is selected from the group consisting of Arg and Lys.

64. The Apelin-13 polypeptide mutant of any one of claims 56 to 63, wherein said aa8’ is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu-2xOEG, carboxy-C 10-24 alkanoyl-γGlu-3xOEG or PEG.

65. The Apelin-13 polypeptide mutant of any one of claims 56 to 64, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

66. The Apelin-13 polypeptide mutant of any one of claims 56 to 65, consisting of a polypeptide represented by general formula (VIII-1) or a polypeptide represented by general formula (VIII-2).

67. The Apelin-13 polypeptide mutant of any one of claims 56 to 66, the polypeptide represented by general formula (VIII-1) is selected from the group consisting of: C 15 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 19 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 21 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 16 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 11 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 13 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 15 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 19 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 21 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 16 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-4xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-5xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-3xOEG-Glu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alk CO-DγGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro- Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-8PEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-2xGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-3xGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); Phenyl-C 17 Ala-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe C 17 Alkyl-CO-Abu-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-Abu-2xGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-Abu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 12 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 14 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 16 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 20 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 22 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-4xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-2xOEG-Glu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-DGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-8PEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-2xGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-3xGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Ala-Glu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-Phe-DMet-Gly-Pro- Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-Abu-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-Abu-2xGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-Abu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-5xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-3xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and COOHC 18 Alkyl-CO-γGlu-OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe.

68. The Apelin-13 polypeptide mutant of any one of claims 56 to 66, the polypeptide represented by general formula (VIII-2) is: C 19 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG3-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG5-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and PEG8-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

69. An Apelin-13 polypeptide mutant comprising (1) a polypeptide represented by general formula (IX): aa9-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-a9-Pro-DMet-Gly-Pro-Phe general formula (IX) wherein a9 is a polar uncharged amino acid, aa9 is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxy-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxy-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues in positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5, n is an integer from 1 to 8; or (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (IX) and having the same function.

70. The Apelin-13 polypeptide mutant of claim 69, wherein the a9 is selected from the group consisting of Ser, Thr, Asn and Gin.

71. The Apelin-13 polypeptide mutant of claim 69 or 70, wherein aa9 is selected from C 11-25 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu-2xOEG, carboxy-C 10-24 alkanoyl-yGlu-3xOEG or PEG.

72. The Apelin-13 polypeptide mutant of any one of claims 69 to 71, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

73. The Apelin-13 polypeptide mutant of any one of claims 69 to 72, consisting of a polypeptide represented by general formula (IX).

74. The Apelin-13 polypeptide mutant of any one of claims 69 to 73, wherein the polypeptide represented by general formula (IX) is: C 19 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 22 alkyl -CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG2-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG3-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); PEG5-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and PEG8-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

75. An Apelin-13 polypeptide mutant comprising (1) a polypeptide represented by general formula (XI-1): aa11-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11-Pro-Phe general formula (XI-1) wherein, a11 is selected from the group consisting of Ala, Phe, Nal, DNal, DMet, Glu or Nle, aa11 is selected from the group consisting of C 11-25 hydrocarbyl-γGlu, carboxy-C 10-24 hydrocarbyl-γGlu, C 11-25 hydrocarbyl-γGlu-mxOEG, carboxy-C 10-24 hydrocarbyl-γGlu-mxOEG, C 11-25 hydrocarbyl-γGlu-nxPEG, carboxy-C 10-24 hydrocarbyl-γGlu-nxPEG or PEG, and the cysteine residues in positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; (2) a polypeptide represented by general formula (XI-2): aa11’-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11’-b11’-Pro-Phe general formula (XI-2) wherein a11' is selected from an amino acid having a hydrophobic side chain or a polar uncharged amino acid, His or Pro, b11' is selected from an amino acid having a hydrophobic side chain or Gly, aa11' is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxy-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxy-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues in positions 1 and 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or (3) a polypeptide having at least 70% homology to the polypeptide represented by general formula (XI-1) or general formula (XI-2) and having the same function.

76. The Apelin-13 polypeptide mutant of claim 75, wherein the a11’ is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gin, His, and Pro.

77. The Apelin-13 polypeptide mutant of claim 75 or 76, wherein the b11’ is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, and Gly.

78. The Apelin-13 polypeptide mutant of any one of claims 75 to 77, wherein each of aa11 and aa11’ is independently selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu-2xOEG, carboxy-C 10-24 alkanoyl-γGlu-3xOEG or PEG.

79. The Apelin-13 polypeptide mutant of any one of claims 75 to 78, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

80. The Apelin-13 polypeptide mutant of any one of claims 75 to 79, consisting of a polypeptide represented by general formula (XI-1) or a polypeptide represented by general formula (XI-2).

81. The Apelin-13 polypeptide mutant of any one of claims 75 to 80, the polypeptide represented by general formula (XI-1) is selected from the group consisting of: C 15 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Nal-Pro-Phe (Disulfide bridge: 1-6).

82. The Apelin-13 polypeptide mutant of any one of claims 75 to 80, the polypeptide of general formula (XI-2) is selected from the group consisting of: C 15 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Nal-Pro-Phe (Disulfide bridge: 1-6); C 19 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6); COOHC 22 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6); PEG2-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6); PEG3-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6); PEG5-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6); and PEG8-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe (Disulfide bridge: 1-6).

83. An Apelin-13 polypeptide mutant comprising (1) a polypeptide of general formula (XII): aa12-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-a12-Pro-Phe General Formula (XII) wherein a12 is selected from an amino acid with a positively charged side chain, an amino acid with a hydrophobic side chain, an amino acid with a polar uncharged side chain or an amino acid with a negatively charged side chain, aa12 is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxy-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxy-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues in positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or (2) a polypeptide having at least 70% homology to the polypeptide of general formula (XII) and having the same function.

84. The Apelin-13 polypeptide mutant of claim 83, wherein the a12 is selected from the group consisting of Arg, Lys, Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gin, Asp, and Glu.

85. The Apelin-13 polypeptide mutant of claim 83 or 84, wherein said aa12 is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu-2xOEG, carboxy-C 10-24 alkanoyl-γGlu-3xOEG or PEG.

86. The Apelin-13 polypeptide mutant of any one of claims 83-85, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

87. The Apelin-13 polypeptide mutant of any one of claims 83 to 86, consisting of a polypeptide of general formula (XII).

88. The Apelin-13 polypeptide mutant of any one of claims 83 to 87, wherein the polypeptide of general formula (XII) is selected from the group consisting of: C 19 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro- Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe (Disulfide bridge: 1-6); C 19 Alkyl-CO-2xγGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly- Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6); C 19 Ala-DArg-DAla-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6); Ac-DLys(2xOEG-γGlu-C 19 Alkyl-CO)-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 1-6); DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 1-6); C 19 Alkyl-CO-γGlu-2xOEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge: 1-6); Ac-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 4-9); Ac-Lys-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 4-9); Ac-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 3-8); Ac-Lys-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 3-8); Ac-Arg-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 3-8); Ac-Lys-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 5-10); Ac-Leu-Arg-Gly-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 5-10); Ac-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2 (Disulfide bridge: 4-9); DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2 (Disulfide bridge: 4-9); COOHC 16 Alkyl-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly- Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge: 4-9); COOHC 18 Alkyl-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly- Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge: 4-9); COOHC 16 Alkyl-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge: 4-9); COOHC 18 Alkyl-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Leu-His-Arg-Gly- Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge: 4-9); COOHC 16 Alkyl-CO-γGlu-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge: 4-9); COOHC 18 Alkyl-CO-γGlu-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge: 4-9); COOHC 18 Alkyl-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly- Pro-Gly-Met-Pro-Phe-NH2 (Disulfide bridge: 4-9); COOHC 16 Alkyl-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His- Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2 (Disulfide bridge: 4-9); COOHC 18 Alkyl-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His- Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2 (Disulfide bridge: 4-9); COOHC 18 Alkyl-CO-γGlu-γGlu-DArg-DLeu-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 5-10); COOHC 18 Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe- NH2(Disulfide bridge: 5-10); COOHC 18 Ala-DArg-DLeu-DArg-DAla-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly- Pro-DMet-DLeu-Pro-Phe-NH2 (Disulfide bridge: 6-11); COOHC 18 Ala-DArg-DAla-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu- Pro-Phe-NH2 (Disulfide bridge: 6-11); and COOHC 18 Ala-DArg-DAla-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Arg-Arg-Gly-Pro-DMet-DLeu- Pro-Phe-NH2(Disulfide bridge: 7-12).

89. An Apelin-13 polypeptide mutant comprising (1) a polypeptide represented by general formula (XIII): aa13-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-a13-Phe General Formula (XIII) wherein, a13 is selected from an amino acid with a hydrophobic side chain, a polar uncharged side chain, a negatively charged side chain or His, aa13 is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxy-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxy-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues in positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5 and n is an integer from 1 to 8; or (2) a polypeptide having at least 70% homology with the polypeptide represented by general formula (XIII) and having the same function.

90. The Apelin-13 polypeptide mutant of claim 89, wherein said a13 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gin, Asp, Glu, and His.

91. The Apelin-13 polypeptide mutant of claim 89 or 90, wherein said aa13 is selected from C 11-25 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu-2xOEG, carboxy-C 10-24 alkanoyl-yGlu-3xOEG or PEG.

92. The Apelin-13 polypeptide mutant of any one of claims 89 to 91, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

93. The Apelin-13 polypeptide mutant of any one of claims 89 to 92, consisting of a polypeptide of Formula (XIII).

94. The Apelin-13 polypeptide mutant of any one of claims 89 to 93, wherein said polypeptide of Formula (XIII) is selected from the group consisting of: C 15 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Tyr- Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-DLeu-Phe (Disulfide bridge: 1-6); COOHC 18 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-DLeu- Phe (Disulfide bridge: 1-6); and COOHC 22 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-DLeu-Phe (Disulfide bridge: 1-6).

95. An Apelin-13 polypeptide mutant comprising (1) a polypeptide of Formula (XIV): aa14-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-a14 Formula (XIV) wherein, a14 is selected from an amino acid with a hydrophobic side chain, a polar uncharged side chain, a negatively charged side chain, His or Nal, a14 is selected from C 11-25 alkanoyl-gamma Glu, carboxy-C 10-24 alkanoyl-gamma Glu, C 11-25 alkanoyl-gamma Glu-mxOEG, carboxy-C 10-24 alkanoyl-gamma Glu-mxOEG, C 11-25 alkanoyl-gamma Glu-nxPEG, carboxy-C 10-24 alkanoyl-gamma Glu-nxPEG or PEG, and the cysteine residues in positions 1, 6 form a disulfide bond, wherein m is an integer from 1 to 5, n is an integer from 1 to 8; or (2) a polypeptide having at least 70% homology to a polypeptide of Formula (XIV) and having the same function.

96. The Apelin-13 polypeptide mutant of claim 95, wherein said a14 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gin, Asp, Glu, His, and Nal.

97. The Apelin-13 polypeptide mutant of claim 95 or 96, wherein said aa14 is selected from C 11-25 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu, carboxy-C 10-24 alkanoyl-yGlu-2xOEG, carboxy-C 10-24 alkanoyl-yGlu-3xOEG or PEG.

98. The Apelin-13 polypeptide mutant of any one of claims 95 to 97, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

99. The Apelin-13 polypeptide mutant of any one of claims 95 to 98, consisting of a polypeptide of Formula (XIV).

100. The Apelin-13 polypeptide mutant of any one of claims 95 to 99, wherein said polypeptide of Formula (XIV) is selected from the group consisting of: C 19 Alkyl-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro- DAsp(Disulfide bridge: 1-6); C 17 alkyl-CO-γGlu-2xOEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro- DTyr (Disulfide bridge: 1-6); and PEG5-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DTyr (Disulfide bridge: 1-6).

101. An Apelin-13 polypeptide mutant comprising (1) a polypeptide of Formula (XV): aa15-Cys-a15-b15-c15-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe Formula (XV) wherein, a15and b15are each independently selected from the group consisting of positively charged side chain amino acids or Pro, c15is selected from the group consisting of a hydrophobic side chain amino acid or a positively charged side chain amino acid, aa15is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxy-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxy-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues in positions 1, 5 form a disulfide bond, wherein m is an integer from 1 to 5, n is an integer from 1 to 8; or (2) a polypeptide having at least 70% homology to a polypeptide of Formula (XV) and having the same function.

102. The Apelin-13 polypeptide mutant of claim 101, wherein said a15 and b15 are each independently selected from the group consisting of Arg, Lys, and Pro.

103. The Apelin-13 polypeptide mutant of claim 101 or 102, wherein c15 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Arg, and Lys.

104. The Apelin-13 polypeptide mutant of any one of claims 101 to 103, wherein said aa15 is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu-2xOEG, carboxy-C 10-24 alkanoyl-γGlu-3xOEG or PEG.

105. The Apelin-13 polypeptide mutant of any one of claims 101 to 104, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

106. The Apelin-13 polypeptide mutant of any one of claims 101 to 105, consisting of a polypeptide of Formula (XV).

107. The Apelin-13 polypeptide mutant of any one of claims 101 to 106, wherein the polypeptide of Formula (XV) is selected from the group consisting of: C 19 Alkyl-CO-γGlu-Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); C 17 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro- Phe (Disulfide bridge: 1-6); and COOHC 22 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro- Phe (Disulfide bridge: 1-6).

108. An Apelin-13 polypeptide mutant comprising (1) a polypeptide of Formula (XVI): aa16-Cys-a16-b16-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe Formula (XVI) wherein, a16 is selected from a positively charged side chain amino acid or Pro, b16 is selected from a hydrophobic side chain amino acid, a positively charged side chain amino acid or Pro, aa16 is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, C 11-25 alkanoyl-γGlu-mxOEG, carboxy-C 10-24 alkanoyl-γGlu-mxOEG, C 11-25 alkanoyl-γGlu-nxPEG, carboxy-C 10-24 alkanoyl-γGlu-nxPEG or PEG, and the cysteine residues in positions 1, 4 form a disulfide bond, wherein m is an integer from 1 to 5, n is an integer from 1 to 8; or (2) a polypeptide having at least 70% homology to the polypeptide of Formula (XVI) and having the same function.

109. The Apelin-13 polypeptide mutant of claim 108, wherein the a16 is selected from the group consisting of Arg, Lys, and Pro.

110. The Apelin-13 polypeptide mutant of claim 108 or 109, wherein b16 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Arg, Lys, and Pro.

111. The Apelin-13 polypeptide mutant of any one of claims 108 to 110, wherein said aa16 is selected from C 11-25 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu, carboxy-C 10-24 alkanoyl-γGlu-2xOEG, carboxy-C 10-24 alkanoyl-γGlu-3xOEG or PEG.

112. The Apelin-13 polypeptide mutant of any one of claims 108 to 111, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

113. The Apelin-13 polypeptide mutant of any one of claims 108 to 112, consisting of a polypeptide of Formula (XVI).

114. The Apelin-13 polypeptide mutant of any one of claims 108 to 113, wherein the polypeptide of Formula (XVI) is selected from the group consisting of: C 19 alkyl-CO-γGlu-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); COOHC 22 Alkyl-CO-γGlu-2xOEG-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and PEG5-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

115. An Apelin-13 polypeptide mutant comprising (1) a polypeptide of Formula (XVII): *-Arg-a17-b17-Leu-*-His-C17-Gly-Pro-DMet-Gly-Pro-Phe Formula (XVII) wherein, a17 and b17 are each independently selected from the group consisting of a positively charged side chain amino acid, or a17 and / or b17 are absent, c17 is a positively charged side chain amino acid, * is a non-natural amino acid, and a cyclic structure is formed between *; or (2) a polypeptide having at least 70% homology to the polypeptide of Formula (XVII) and having the same function.

116. The Apelin-13 polypeptide mutant of claim 115, wherein the a17 and b17 are each independently selected from the group consisting of Arg, Lys, and Pro.

117. The Apelin-13 polypeptide mutant of claim 115, wherein the a17 and b17 are both absent.

118. The Apelin-13 polypeptide mutant of any one of claims 115 to 117, wherein b16 is selected from the group consisting of Arg and Lys.

119. The Apelin-13 polypeptide mutant of any one of claims 115 to 118, wherein * is selected from 120. The Apelin-13 polypeptide mutant of any one of claims 115 to 119, wherein the cyclic structure formed between * is a staple isopeptide structure.

121. The Apelin-13 polypeptide mutant of any one of claims 115 to 120, wherein the * form a cyclic structure is 122. The Apelin-13 polypeptide mutant of any one of claims 115 to 121, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

123. The Apelin-13 polypeptide mutant of any one of claims 115 to 122, consisting of a polypeptide according to general formula (XVII).

124. The Apelin-13 polypeptide mutant of any one of claims 115 to 123, wherein the polypeptide of Formula (XVII) is selected from:

125. An Apelin-13 polypeptide mutant comprising (1) a polypeptide according to general formula (XVIII): a18-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-b18-Pro-Phe General formula (XVIII) wherein a18 is selected from: MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGA MKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGA MKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLGGAGGAGGAGGAGGA MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGARLR MKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGARLR MKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLGGAGGAGGAGGAGGARLR b18 is selected from an amino acid having a hydrophobic side chain or an amino acid having a negatively charged side chain, and the cysteine residues at positions 1 and 6 form a disulfide bond; or (2) a polypeptide having at least 70% homology to a polypeptide represented by general formula (XVIII) and having the same function.

126. The Apelin-13 polypeptide mutant of claim 125, wherein the b18 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Asp, and Glu.

127. The Apelin-13 polypeptide mutant of claim 125 or 126, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

128. The Apelin-13 polypeptide mutant of any one of claims 125 to 127, consisting of a polypeptide represented by general formula (XVIII).

129. The Apelin-13 polypeptide mutant of any one of claims 125 to 128, wherein the polypeptide of Formula (XVIII) is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO:

8.

130. An Apelin-13 polypeptide mutant comprising (1) a polypeptide of Formula (XIX): a19-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-b19-c19-Pro-Phe Formula (XIX) wherein, a19 is selected from the group consisting of: MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGA MKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGA MKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLGGAGGAGGAGGAGGA MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGARLR MKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGARLR MKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLGGAGGAGGAGGAGGARLR b19 is selected from an amino acid having a hydrophobic side chain or an amino acid having a negatively charged side chain, c19 is selected as an amino acid having a hydrophobic side chain, and the cysteine residues at positions 1 and 6 form a disulfide bond; or (2) a polypeptide having at least 70% homology to a polypeptide represented by general formula (XIX) and having the same function.

131. The Apelin-13 polypeptide mutant of claim 130, wherein b19 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, and Trp.

132. The Apelin-13 polypeptide mutant of claim 130 or 131, wherein c19 is selected from the group consisting of Ala, Val, lie, Leu, Met, Phe, Tyr, Trp, Asp, and Glu.

133. The Apelin-13 polypeptide mutant of any one of claims 130-132, wherein the Apelin-13 polypeptide mutant binds to M183 ECL2 , F110 3.33 , I109 3.32 , K268 6.55 , Y264 6.51 , K265 6.55 , Y35 1.39 , T295 7.39 , Y299 7.49 , F78 2.53 , W85 2.60 , Y88 2.63 , Y93 ECL1 , M113 3.36 , R168 4.64 , Y185 ECL2 , E198 5.35 , Y271 6.58 , S275 6.62 , M288 7.32 , F291 7.35 , P163 4.59 , L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 , or Y182 ECL2 .

134. The Apelin-13 polypeptide mutant of any one of claims 130 to 133, consisting of a polypeptide according to general formula (XIX).

135. The Apelin-13 polypeptide mutant of any one of claims 130 to 134, wherein the polypeptide according to general formula (XIX) is selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO:

5.

136. A pharmaceutical composition comprising an Apelin-13 polypeptide mutant of any one of claims 1 to 135, and a pharmaceutically acceptable carrier, diluent or excipient.

137. The pharmaceutical composition of claim 136, further comprising at least another active ingredient.

138. The pharmaceutical composition of claim 137, wherein the at least another active ingredient is capable of acting on GLP1R, GLP2R, GIPR, GCGR, FGF21R, NPY2R or GDF15.

139. The pharmaceutical composition of claim 137 or 138, wherein the at least another active ingredient is selected from the group consisting of Vurolenatide, exenatide, ROSE-010, GX-G6, orforglipron, FNP-120, UTMD-GLP-1, GLP-1 mimetics, VTC-G15, curaglutide, Exendin-4, P-11, CTRS-101, HD-7671, HTL-097, PGN-OB2, GL-0034, dulaglutide, semaglutide, enagoglutide, supalutai, rE-4, HS-20004, BPI-3016, ZT002, ECC-5004, TJ-103, SHR-2042, MDR-001, LPXT-007, Tirzepatide, CT-868, NNC-0090-2746, AMG133, CT-388, LY3493269, VK-2735, DA4-JC, HISHS-3001, HISHS-2001, LBT-6030, SCO-094, HS-20094, GMA106, HZ010, BGM0504, HZ012, XW003+XW017, THDBH120, BEBT-808, RAY-1225, Cotadutide, Pegapamodutide, BI-456906, Mazdutide / IBI362, efinopegdutide, Pemvidutide, NN-6177, ZP-2929, oxyntomodulin, G-49, AGM-217, JNJ-54728518, DA-1726, HM-14220, DD01, PB-718, TB001, SHR-1816, TB-001, YH-25724, HEC88473, AP-026, GLP-1-Fc-PYY, ZP-7570 (dapiglutide), HTL-30023, QL-1005, LY3437943 (retatrutide), HM15211, SAR441255, HISHS-3001, MWN-101, and DR10624.

140. A method of treating or preventing a disease or condition responsive to agonism of the APLNR receptor, comprising administering to an individual in need of the method a therapeutically or prophylactically effective amount of the Apelin-13 polypeptide mutant of any one of claims 1 to 135 or the pharmaceutical composition of any one of claims 136 to 139.

141. The method of claim 140, wherein the disease or condition is selected from the group consisting of maintenance of muscle mass, fat loss, kidney disease, muscle building, muscle atrophy, Brugada syndrome, water retention, diabetes, obesity, traumatic brain injury, amyotrophic lateral sclerosis, burns, preeclampsia, anti-aging, pain relief, sleep apnea syndrome, pulmonary embolism, asthma, stress-induced gastric ulcer, liver fibrosis, lung injury, brain injury, ischemic stroke, cancer, Alzheimer's disease, Parkinson's disease, stroke, and neurodegenerative disease.

142. The method of claim 140 or 141, wherein the individual is a mammal, preferably a human.

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